Method for generating Retinal Pigment Epithelium (RPE) cells from Induced Pluripotent Stem Cells (IPSCs)

ABSTRACT

High efficiency methods for producing retinal pigment epithelial cells from induced pluripotent stem cells (iPSCs) are disclosed herein. The iPSCs are produced from somatic cells, including retinal pigment epithelial (RPE) cells, such as fetal RPE stem cells. In some embodiments, the iPSC include a tyrosinase promoter operably linked to a marker. Methods are disclosed for using the RPE cells, such as for treatment. Methods for screening for agents that affect RPE differentiation are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. patent application Ser. No. 14/764,959,filed on Jul. 30, 2015, which is the U.S. National Stage ofInternational Application No. PCT/US2014/014160, filed Jan. 31, 2014,which was published in English under PCT Article 21(2), which claims thebenefit of U.S. Patent Application No. 61/759,988, filed Feb. 1, 2013.The prior applications are incorporated herein by reference in theirentirety.

FIELD OF THE DISCLOSURE

This disclosure is related to the field of retinal pigment epithelium(RPE) cells, and specifically to methods for producing RPE cells fromstem cells.

BACKGROUND

The retina is a layer of specialized light sensitive neural tissuelocated at the inner surface of the eye of vertebrates. Light reachingthe retina after passing the cornea, the lens and the vitreous humor istransformed into chemical and electrical events that trigger nerveimpulses. The cells that are responsible for transduction, the processfor converting light into these biological processes are specializedneurons called photoreceptor cells.

The retinal pigment epithelium (RPE) is a polarized monolayer of denselypacked hexagonal cells in the mammalian eye that separates the neuralretina from the choroid. The cells in the RPE contain pigment granulesand perform a crucial role in retinal physiology by forming ablood-retinal barrier and closely interacting with photoreceptors tomaintain visual function by absorbing the light energy focused by thelens on the retina. These cells also transport ions, water, andmetabolic end products from the subretinal space to the blood and takeup nutrients such as glucose, retinol, and fatty acids from the bloodand deliver these nutrients to photoreceptors.

RPE cells are also part of the visual cycle of retinal: Sincephotoreceptors are unable to reisomerize all-trans-retinal, which isformed after photon absorption, back into 11-cis-retinal, retinal istransported to the RPE where it is reisomerized to 11-cis-retinal andtransported back to the photoreceptors.

Many ophthalmic diseases, such as (age-related) macular degeneration,macular dystrophies such as Stargardt's and Stargardt's-like disease,Best disease (vitelliform macular dystrophy), and adult vitelliformdystrophy or subtypes of retinitis pigmentosa, are associated with adegeneration or deterioration of the retina itself or of the RPE. It hasbeen demonstrated in animal models that photoreceptor rescue andpreservation of visual function could be achieved by subretinaltransplantation of RPE cells (Coffey et al. Nat. Neurosci. 2002:5,53-56; Lin et al. Curr. Eye Res. 1996:15, 1069-1077; Little et al.Invest. Ophthalmol. Vis. Sci. 1996:37, 204-211; Sauve et al.Neuroscience 2002:114, 389-401). There is a need to find ways to produceRPE cells, such as from human stem cells, that can be used for thetreatment of retinal degenerative diseases and injuries.

SUMMARY OF THE DISCLOSURE

High efficiency methods for producing retinal pigment epithelial cellsfrom induced pluripotent stem cells (iPSCs) are disclosed herein. TheiPSCs are produced from somatic cells, including retinal pigmentepithelial cells, such as fetal retinal pigment epithelial stem cells.

In some embodiments, the method for producing human retinal pigmentepithelial cells includes producing embryoid bodies from human inducedpluripotent stem cells. The embryoid bodies are cultured in a firstmedium comprising two Wnt pathway inhibitors and a Nodal pathwayinhibitor. The embryoid bodies are plated on a tissue culture substratein a second medium. In certain embodiments, the second medium (a) doesnot include basic fibroblast growth factor (bFGF) (b) includes a basicfibroblast growth factor (bFGF) inhibitor, the two Wnt pathwayinhibitors, and the Nodal pathway inhibitor; (c) includes about 20 toabout 90 ng of Noggin; and (d) includes about 1 to about 3% knock outserum replacement to form differentiating retinal pigment epithelialcells. The differentiating retinal pigment epithelial cells are thencultured in a third medium comprising ACTIVAN A and WNT3a. The cells arethen cultured in retinal pigment epithelial cell (RPE) medium thatincludes about 5% fetal serum, a canonical WNT inhibitor, anon-canonical WNT inducer, and inhibitors of the Sonic and FGF pathwayto produce human retinal pigment epithelial cells.

In additional embodiments, methods are disclosed for detecting RPEcells, or confirming that a cell is a RPE cell. In yet otherembodiments, methods are disclosed for determining if a test agentaffects the production of RPE cells from an iPSC. In furtherembodiments, methods are provided to identify a test agent thatincreases gene expression and can be used as a therapeutic agent. In yetother embodiments, methods are provided to identify a test agent thatincreases RPE survival in response to a proteotoxic insult or stress.

The foregoing and other features and advantages of the invention willbecome more apparent from the following detailed description of aseveral embodiments, which proceeds with reference to the accompanyingfigures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic diagram of the RPE-specific construct, whichincludes a constitutive promoter operably linked to a marker (forexample, red fluorescent protein, RFP) and the RPE-specific tyrosinasepromoter operably linked to a second marker (for example, greenfluorescent protein, GFP). Digital images of the results achieved arealso shown. The RPE-specific promoter/enhancer-GFP construct was firsttested in a transgenic mouse. RPE-specific GFP expression (left image)was achieved. This construct was then combined with a constitutive RFPand transduced into iPSCs. At the iPSC stage only RFP is expressed(right image). This construct can be used to produce an iPSC lineexpressing RPE specific GFP.

FIG. 2 is a schematic diagram of the steps in differentiation of RPEfrom human iPSCs. The protocol was optimized to obtain RPE cells withhigh efficiency, while inhibiting other lineages. Markers for thedifferent stages are shown.

FIG. 3 is a schematic diagram and digital images of RPE-Specific GFPExpression in the Reporter iPSC line. GFP positive cells appear whencells reach committed RPE stage, which begins 4 weeks afterdifferentiation begins. GFP positive cells acquire epithelial characterand begin to pigment in one more week. The expression of reporter after28 and 35 days in culture is shown.

FIG. 4 is a table of results from FACS purified GFP positive cells,showing the cells are enriched for expression of early RPE genes. GFPpositive cells and negative cells from the same dish were purified byFACS and the expression of RPE-specific markers was compared in the twopopulations. GFP positive cells are several fold enriched in theexpression of RPE-specific genes.

FIGS. 5A to 5B are a set of digital images showing FACS purified GFPpositive cells differentiate into pigmented RPE cells. When FACSpurified GFP positive and negative cells were re-seeded on to cultureplates, only GFP positive cells grew to form homogenous pigmentedcultures. This demonstrated that GFP expression truly represents RPEfate in this iPSC line

FIG. 6 is a set of digital images showing embryoid body (EB) sizeaffects RPE differentiation. Differences in the number of pigmentedcells obtained using varying size EBs are shown.

FIG. 7 is a set of digital images showing embryoid body (EB) numberaffects RPE differentiation. FACS analysis shows that the number of GFPpositive cells in the sixth week of the differentiation process. Thenumber of GFP positive cells changes as the number of EBs plated perwell of the culture plate change.

FIGS. 8-10 are tables showing the fold change in PAX6 expression withdifferent culture conditions.

FIGS. 11-13 are tables showing the fold change in MITF expression usingdifferent culture conditions. MITF expression should start to increasearound week 3 of differentiation, when the cells are getting to theoptic vesicle stage.

FIG. 14 is a table showing the percent GFP positive cells (representingcells of the RPE fate) using different culture conditions.

FIG. 15 is a set of digital images showing the differentiation of iPSCinto RPE. The cells were cultured using the media and protocols listedin Example 1. For these studies, the KO serum was the only componentthat was varied.

FIG. 16 is a set of digital images showing that canonical WNT and WNT3aimproves the efficiency of production of RPE from iPSCs. WNT3A is acanonical WNT signaling regulator. The digital images show pigmentedcolonies in each dish after eight weeks of culture. For theseexperiments MEDIUM 2 (FGF2−0.5 ng/ml)+MEDIUM 3 (NA or NAW), whereinNA=Nicotinamide & ACTIVIN, NAW=Nicotinamide, ACTIVIN & WNT3a. bFGF(FGF2) was included 0.5 ng/ml in medium 2.

FIG. 17 is a set of digital images and plots showing WNT3a significantlyincreases the number of GFP positive cells in differentiating iPSCcultures. The upper panel shows GFP positive and RFP positive cells in adish. The lower panel shows FACS analysis for the GFP signal. For theseexperiments, MEDIUM 2 (FGF2−0.5 ng/ml)+MEDIUM 3 (NA or NAW), whereinNA=Nicotinamide & ACTIVIN, NAW=Nicotinamide, ACTIVIN & WNT3a.

FIG. 18 is a set of digital images and plots showing the use of a GFPreporter to optimize iPSC to RPE Differentiation. Culture in the thirdmedium significantly improved the production of RPE cells, as evidenceby GFP expression and production of pigment, after 6 weeks.

Medium 2—NO FGF2+PD 0325901 (10 mM)+Noggin (50 ng/ul)+DKK1

Medium 3—Activin A (150 ng/ml)+WNT3a (100 ng/ml)

Medium 4—See the examples section below.

FIG. 19 is a digital image showing that WNT3a generates RPE cells withhomogeneous pigmentation. Cells from these dishes were trypsinized andreplated into T-25 flasks. For these experiments, MEDIUM 2 (FGF2−0.5ng/ml)+MEDIUM 3 (NA or NAW), wherein NA=Nicotinamide & ACTIVIN,NAW=Nicotinamide, ACTIVIN & WNT3a.]

FIG. 20 is a digital image of iPSC-derived RPE, NAW treated (92% GFPpositive). At higher resolution these cells look quite homogenous interms of RPE morphology, GFP expression, and pigmentation.

FIG. 21 is a table showing that inhibition of endogenous pathways (WNT,FGF, SONIC) in iPSC-RPE produces fully-mature RPE cultures Medium #4−5%RPE medium+inhibitors of WNT, FGF, SONIC. WNT inhibitors include DKK1and non-canonical WNT (WNT5a), FGF inhibitor SU5042, and SONIC inhibitorCyclopamine.

FIG. 22 is a set of graphs and a Venn diagram illustrating RPE geneexpression is iPSC-RPE. The expression of a total 384 genes wasevaluated (Strunnikova et al., Hum Mol Genet. 2010 Jun. 15;19(12):2468-86). The top 350 genes were selected; 34 genes werecontrols. A custom 384-well plate was developed to analyze theexpression of all these genes by real-time PCR. This plate/array can beused to authenticate iPSC-RPE cells. They include RPE gene signature,fetal-RPE enriched genes, and adult-RPE enriched genes. A comparison of˜100 fetal-RPE enriched genes among three iPSC lines derived RPE isshown.

FIG. 23 is a schematic diagram and graphs evidencing the physiology ofiPSC derived RPE. Selective CO₂ permeability of the apical surface ofRPE cells is shown (Adijanto et al., J Gen Physiol. 2009 June;133(6):603-22). Photoreceptors secrete CO₂ towards the apical surface ofRPE cells. Therefore, this surface has been evolutionarily selected totrap CO₂. This function can be measured in vitro by changing CO₂concentrations in the apical or the basal baths of RPE cells growing intranswells. If RPE cells trap CO₂, they respond by a reduction in pH,which can be measured by a ratio-metric dye. iPSC-derived RPE functionsimilar to the native RPE for differential CO₂ uptake only on the apicalside.

FIG. 24 is a set of graphs showing changes in electrophysiologicalproperties of iPSC-derived RPE cells by changes in extracellular ionicconcentrations. When light hits the photoreceptors, they depolarize byshutting down their potassium (K) channels. This reduces theconcentration of K-ions in between photoreceptors and RPE (subretinalspace) from 5 mM to 1 mM. RPE cells respond to this changingconcentration by hyperpolarizing and opening their K channels toincrease the subretinal K concentration back to 5 mM.

FIGS. 25A-25D are schematic diagrams, digital images and graphs. (A)Schematic of the step-wise protocol for differentiation of iPSCs intoRPE, authentication of RPE cells, and their use for a multiplexscreening. The different type of media used throughout the process andthe timeline of the process are shown. (B) Characterization of the iPSCline used for generating RPE for screening. Fully confluent iPSCs werestained with antibodies against indicated pluripotency markers. Cellsexpress high levels of all these markers. Bright field images show iPSCcolony morphology. (C) qRT-PCR analysis of pluripotency markers NANOG,OCT4, SOX2, c-MYC, and KLF4 in iPSC line used for RPE differentiation.Fibroblasts and embryonic stem cell RNA are used for comparison. (D)Immunostaining of iPSCs spontaneously differentiated in vitro after bFGFwithdrawal. iPSCs are able to generate cells from the three germ layers(AFP=endoderm; TUJ1=ectoderm; SMA=mesoderm). The bar represents 50microns. KSR—knockout serum replacement containing medium,NIC—nicotinamide.

FIGS. 26A-26D show authentication of iPSC-derived RPE. RPE cells weregrown on semi-permeable transwells until fully confluent andcharacterized by immunostaining for RPE markers (A), electron microscopy(B), intracellular calcium responses (C), electrophysiological responses(D). (A) RPE cells were stained with antibodies against EZRIN, DCT,SLC16A1, CLCN2 (grey) and ZO1 (bright lines). (B) Electron micrograph ofa section of RPE growing on a semi-permeable transwell. Cells showseveral features typical of RPE including extensive apical process,apically localized pigmented melanosomes (me), and tight-junctions (tj)between adjoining cells. (C) Similar to primary fetal RPE cells,iPSC-derived RPE show a baseline calcium concentration of 110 nM.Addition of ATP to the apical bath activates apical P2Y2 receptorsresulting in a spike in intracellular calcium concentration. (D) Apicaland basal membrane resting potentials were measured in response tochanging potassium (K) and ATP concentrations in the apical bath.Similar to the primary cultures of human RPE, these cells hyperpolarizewhen K concentration is reduced to 1 mM and depolarize when ATP is addedto the apical bath.

FIGS. 27A-27C show a schematic of the multiplex assay performed in a96/384-well plate format. (A) Brief summary and the time line for themultiplex assay performed using iPSC-derived RPE. Assay can be performedin a day and a half with full automation. (B) Magnetic beads labeledwith a unique fluorophores capture specific mRNAs through anti-senseoligo-nucleotide interactions of capture probe (CP) and multiple captureextenders (CEs). CE is a branched oligo-nucleotide; one side of it isanti-sense to CP and the other side has variable sequence that itanti-sense to the specific mRNA. Label extender (LE) also a branchedoligo-nucleotide that binds to different regions of the mRNA and allowsthe binding of pre-amplifier and amplifier oligos through anti-senseinteractions. Biotin containing label probes and streptavidin-conjugatedphycoerythrin (SAPE) bind to amplifier oligos and are detected using theLuminex flow reader. (C) Table summarizes mRNA detected in this assay,their accession numbers, length, and the region where capture extendersand label extenders bind.

FIGS. 28A-28H show proof of principle for multiplex gene expressionassay in 96-well plates. (A-D) Bright field images of iPSCs and RPEcells growing in a 96-well plate. (A) iPSCs (28B, C) iPSC-derived RPEseeded at two different cell densities (25,000 cells/well and 50,000cells/well) (D) Primary human fetal RPE (50,000 cells/well). (E-H)Results obtained in the multiplex assay. (E, G) Expression of indicatedgenes was normalized to geomean of HPRT1 and B2M genes. Results are showas fold change in gene expression in iPSC-derived RPE seeded at lowercell density (white bar) and seeded at higher cell density (black bar)normalized either to undifferentiated iPSCs (28E) or to the primaryfetal RPE (G). (F, H) Pearson's correlation analysis between geneexpression results obtained from the qRT-PCR assay and the resultsobtained from the multiplex gene expression assay performed in 96-wellplates. The correlation is significant for iPSCs (r=0.687) and highlysignificant for primary RPE cells (r=0.751).

FIG. 29 is a bar graph showing the sensitivity of iPSC-derived RPE cellsto various proteotoxic stressors. RPE cells differentiated from thereporter iPS cell line were grown in 384-well plates and treated withindicated stressors (conc. 10 μM). GRP signals were measured four daysafter the treatment. Thapsigargin, A23187, and 2-Deoxy-D-Glucoseaffected RPE phenotype.

FIG. 30 is a digital image showing that aphidicolin treatment generatespolarized retinal pigment epithelia cells. The retinal pigmentepithelial cells treated with aphidicolon treatment have more extensiveapical process as compared to non-treated cells.

FIG. 31 is a set of graphs showing that aphidicolin treatment improvesthe function of iPSC derived retinal pigment epithelial cells. RPE cellstreated with aphidicolin have higher transepithelial resistance,membrane potential, and show better physiological responses such as theability to hyperpolarize in response to low potassium concentration orATP application on the apical side.

FIGS. 32A-32D are digital images showing alginate coating enhances cellviability and reduces cell detachment during cryopreservation. Celldeath assessed by ethidium homodimer-1 staining (grey). (A) Monolayercryopreserved without alginate treatment using CRYOSTOR® CS10 solution.(B) Monolayer cryopreserved with alginate coating using CRYOSTOR® CS10solution. (C) Monolayer cryopreserved without alginate coating using 10%DMSO media. (D) Monolayer cryopreserved with alginate coating using 10%DMSO media.

FIG. 33 is a set of digital images showing the protocol for growingiPSC-derived RPE on an artificial biodegradable scaffold using aSNAPWELL™ system.

FIGS. 34A-34B are digital images showing pigmentation following cultureof iPSCs in 10% (A) or 1.5% KOSR in RDM for the first 3 weeks of thedifferentiation protocol and photographed after 16 weeks in culture.

FIGS. 35A-35H show the proof of principle for multiplex gene expressionassay in 384-well plates. Bright field images of iPSC-derived RPE (A, B)and human fetal RPE (C, D) seeded at two different cell densitiesgrowing in 384-well plates. (E, G) Fold change in gene expression iniPSC-derived RPE seeded at lower cell density (white bar) and seeded athigher cell density (black bar) normalized to the higher cell density ofprimary fetal RPE. Expression of indicated genes was normalized togeomean of HPRT1 and B2M housekeeping genes. Almost identical resultswere obtained with high bead number (E) and low bead number (G).Pearson's correlation analysis shows a very high correlation betweenresults obtained from the qRT-PCR assay and the results obtained from384-well multiplex gene expression assay. The coefficient values arer=0.908 and r=0.891 respectively for different number of beads.

FIGS. 36A-36F show the detection range of probes used for the multiplexassay. Four fold serial dilution of (A) purified human RPE, (B) iPSCs,(C) iPSC-derived RPE (low density), (D) iPSC-derived RPE (high density),(E) primary fetal RPE (low density), and (F) primary fetal RPE (highdensity) were used to determine range of detection of probe sets usedfor the multiplex assay. R2 value for each probe was more than 0.9suggesting a linear range of detection over 16-fold dilution of thelysates.

SEQUENCE LISTING

The Sequence Listing is submitted as an ASCII text file4239-89784-16_Sequence_Listing.txt, May 2, 2018, 8.05 KB], which isincorporated by reference herein.

The nucleic and amino acid sequences are shown using standard letterabbreviations for nucleotide bases, and one or three letter code foramino acids, as defined in 37 C.F.R. 1.822. Only one strand of eachnucleic acid sequence is shown, but the complementary strand isunderstood as included by any reference to the displayed strand.

SEQ ID NO: 1 is the nucleic acid sequence of an exemplary humantyrosinase enhancer.

SEQ ID NO: 2 is an exemplary promoter sequence.

DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS

High efficiency methods for producing retinal pigment epithelial cellsfrom induced pluripotent stem cells (iPSCs) are disclosed herein. TheiPSCs are produced from somatic cells, including retinal pigmentepithelial cells, such as fetal retinal pigment epithelial stem cells.In some embodiments, the cells are human.

The methods disclosed herein are used to produce large numbers ofdifferentiated RPE cells for use in screening assays, to study the basicbiology of the RPE, and as therapeutics. The RPE cells can include atyrosinase enhancer operably linked to a nucleic acid encoding a marker.In some embodiments, RPE cells produced using the methods disclosedherein can be formulated and used to treat retinal degenerativediseases. Thus, compositions are disclosed that include RPE cells,including substantially purified preparations of RPE cells. Screeningassays are also disclosed for the identification of agents that modulateRPE cell proliferation and/or alter RPE cell differentiation. Agentsidentified using such screening assays may be used in vitro or in vivo.

Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of thisdisclosure, the following explanations of specific terms are provided:

Activin: Members of the transforming growth factor beta (TGF-beta)superfamily which participate in regulation of several biologicalprocesses, including cell differentiation and proliferation. Activin Ais a member of this family that mediates its biological effects througha complex of transmembrane receptor serine/threonine kinases, and bindsto specific Activin A receptors. It is a dimer composed of two subunits.Activin A participates in regulation of stem cell maintenance, viaSMAD-dependent activation transcription of marker of pluripotency likePOU class 5 homeobox 1 (Oct-3/4), nanog, nodal, and nodal-signalingregulators, Left-right determination factor 1 and 2 (Lefty-B andLefty-A). Activin A also stimulates transcription of several hormonessuch as Gonadotropin-releasing hormone. An exemplary sequence forActivin A is provided in GENBANK® Accession No. NM_002192.

Agent: Any protein, nucleic acid molecule (including chemically modifiednucleic acids), compound, small molecule, organic compound, inorganiccompound, or other molecule of interest. Agent can include a therapeuticagent, a diagnostic agent or a pharmaceutical agent. A therapeutic orpharmaceutical agent is one that alone or together with an additionalcompound induces the desired response (such as inducing a therapeutic orprophylactic effect when administered to a subject).

Agonist or Inducer: An agent that binds to a receptor of a cell or aligand of such a receptor and triggers a response by that cell, oftenmimicking the action of a naturally occurring substance. In oneembodiment, a Frizzled (Fzd) agonist binds to a Fzd receptor andpotentiates or enhances the Wnt/beta-catenin signaling pathway.

Alter: A change in an effective amount of a substance or parameter ofinterest, such as a polynucleotide, polypeptide or a property of a cell.An alteration in polypeptide or polynucleotide or enzymatic activity canaffect a physiological property of a cell, such as the differentiation,proliferation, or senescence of the cell. The amount of the substancecan be changed by a difference in the amount of the substance produced,by a difference in the amount of the substance that has a desiredfunction, or by a difference in the activation of the substance. Thechange can be an increase or a decrease. The alteration can be in vivoor in vitro. In several embodiments, altering is at least about a 50%,60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% increase ordecrease in the effective amount (level) of a substance, theproliferation and/or survival of a cells, or the activity of a protein,such as an enzyme.

Amplification: of a nucleic acid molecule (e.g., a DNA or RNA molecule)refers to use of a technique that increases the number of copies of anucleic acid molecule in a sample. An example of amplification ispolymerase chain reaction (PCR), in which a sample is contacted with apair of oligonucleotide primers under conditions that allow for thehybridization of the primers to a nucleic acid template in the sample.The primers are extended under suitable conditions, dissociated from thetemplate, re-annealed, extended, and dissociated to amplify the numberof copies of the nucleic acid. The product of amplification can becharacterized by electrophoresis, restriction endonuclease cleavagepatterns, oligonucleotide hybridization or ligation, and/or nucleic acidsequencing using standard techniques.

Other examples of amplification include quantitative real-timepolymerase chain reaction (qPCR), strand displacement amplification, asdisclosed in U.S. Pat. No. 5,744,311; transcription-free isothermalamplification, as disclosed in U.S. Pat. No. 6,033,881; repair chainreaction amplification, as disclosed in PCT publication WO 90/01069;ligase chain reaction amplification, as disclosed in European patentpublication EP-A-320,308; gap filling ligase chain reactionamplification, as disclosed in U.S. Pat. No. 5,427,930; and NASBA RNAtranscription-free amplification, as disclosed in U.S. Pat. No.6,025,134.

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects, for example, non-human primates, dogs,cats, horses, rabbits, pigs, mice, rats, and cows.

Antagonist or Inhibitor: An agent that blocks or dampens a biochemicalor biological response when bound to a receptor or a ligand of thereceptor. Antagonists mediate their effects through receptorinteractions by preventing agonist-induced responses. In one embodiment,a Frizzled (Fzd) antagonist binds to a Fzd receptor or to a Fzd ligand(such as Wnt) and inhibits the Wnt/beta-catenin signaling pathway.

Antibody: A polypeptide ligand comprising at least a light chain orheavy chain immunoglobulin variable region which specifically recognizesand binds an epitope of an antigen. Antibodies are composed of a heavyand a light chain, each of which has a variable region, termed thevariable heavy (V_(H)) region and the variable light (V_(L)) region.Together, the V_(H) region and the V_(L) region are responsible forbinding the antigen recognized by the antibody.

Antibodies include intact immunoglobulins and the variants and portionsof antibodies well known in the art, such as Fab fragments, Fab′fragments, F(ab)′₂ fragments, single chain Fv proteins (“scFv”), anddisulfide stabilized Fv proteins (“dsFv”). A scFv protein is a fusionprotein in which a light chain variable region of an immunoglobulin anda heavy chain variable region of an immunoglobulin are bound by alinker, while in dsFvs, the chains have been mutated to introduce adisulfide bond to stabilize the association of the chains. The term alsoincludes genetically engineered forms such as chimeric antibodies (forexample, humanized murine antibodies), heteroconjugate antibodies (suchas, bispecific antibodies). See also, Pierce Catalog and Handbook,1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J., Immunology,3^(rd) Ed., W. H. Freeman & Co., New York, 1997.

Typically, a naturally occurring immunoglobulin has heavy (H) chains andlight (L) chains interconnected by disulfide bonds. There are two typesof light chain, lambda (λ) and kappa (k). There are five main heavychain classes (or isotypes) which determine the functional activity ofan antibody molecule: IgM, IgD, IgG, IgA and IgE.

Differentiation: Refers to the process whereby relatively unspecializedcells (such as embryonic stem cells or other stem cells) acquirespecialized structural and/or functional features characteristic ofmature cells. Similarly, “differentiate” refers to this process.Typically, during differentiation, cellular structure alters andtissue-specific proteins appear.

Embryoid Bodies: Three-dimensional aggregates of pluripotent stem cells.These cells can undergo differentiation into cells of the endoderm,mesoderm and ectoderm. In contrast to monolayer cultures, the spheroidstructures that are formed when pluripotent stem cells aggregate enablesthe non-adherent culture of EBs in suspension, which is useful forbioprocessing approaches. The three-dimensional structure, including theestablishment of complex cell adhesions and paracrine signaling withinthe EB microenvironment, enables differentiation and morphogenesis.

Embryo: A cellular mass obtained by one or more divisions of a zygote oran activated oocyte with an artificially reprogrammed nucleus withoutregard to whether it has been implanted into a female. A “morula” is thepreimplantation embryo 3-4 days after fertilization, when it is a solidmass, generally composed of 12-32 cells (blastomeres). A “blastocyst”refers to a preimplantation embryo in placental mammals (about 3 daysafter fertilization in the mouse, about 5 days after fertilization inhumans) of about 30-150 cells. The blastocyst stage follows the morulastage, and can be distinguished by its unique morphology. The blastocystis generally a sphere made up of a layer of cells (the trophectoderm), afluid-filled cavity (the blastocoel or blastocyst cavity), and a clusterof cells on the interior (the inner cell mass, ICM). The ICM, consistingof undifferentiated cells, gives rise to what will become the fetus ifthe blastocyst is implanted in a uterus.

Embryonic stem cells: Embryonic cells derived from the inner cell massof blastocysts or morulae, optionally that have been serially passagedas cell lines. The term includes cells isolated from one or moreblastomeres of an embryo, preferably without destroying the remainder ofthe embryo. The term also includes cells produced by somatic cellnuclear transfer. “Human embryonic stem cells” (hES cells) includesembryonic cells derived from the inner cell mass of human blastocysts ormorulae, optionally that have been serially passaged as cell lines. ThehES cells may be derived from fertilization of an egg cell with sperm orDNA, nuclear transfer, parthenogenesis, or by means to generate hEScells with homozygosity in the HLA region. Human ES cells can beproduced or derived from a zygote, blastomeres, or blastocyst-stagedmammalian embryo produced by the fusion of a sperm and egg cell, nucleartransfer, parthenogenesis, or the reprogramming of chromatin andsubsequent incorporation of the reprogrammed chromatin into a plasmamembrane to produce an embryonic cell. Human embryonic stem cellsinclude, but are not limited to, MAO1, MAO9, ACT-4, No. 3, H1, H7, H9,H14 and ACT30 embryonic stem cells. Human embryonic stem cells,regardless of their source or the particular method use to produce them,can be identified based on (i) the ability to differentiate into cellsof all three germ layers, (ii) expression of at least Oct-4 and alkalinephosphatase, and (iii) ability to produce teratomas when transplantedinto immunocompromised animals.

Expand: A process by which the number or amount of cells in a cellculture is increased due to cell division. Similarly, the terms“expansion” or “expanded” refers to this process. The terms“proliferate,” “proliferation” or “proliferated” may be usedinterchangeably with the words “expand,” “expansion”, or “expanded.”Typically, during an expansion phase, the cells do not differentiate toform mature cells, but divide to form more cells.

Expression: The process by which the coded information of a gene isconverted into an operational, non-operational, or structural part of acell, such as the synthesis of a protein. Gene expression can beinfluenced by external signals. For instance, exposure of a cell to ahormone may stimulate expression of a hormone-induced gene. Differenttypes of cells can respond differently to an identical signal.Expression of a gene also can be regulated anywhere in the pathway fromDNA to RNA to protein. Regulation can include controls on transcription,translation, RNA transport and processing, degradation of intermediarymolecules such as mRNA, or through activation, inactivation,compartmentalization or degradation of specific protein molecules afterthey are produced.

Feeder layer: Non-proliferating cells (such as irradiated cells) thatcan be used to support proliferation of stem cells. Protocols for theproduction of feeder layers are known in the art, and are available onthe internet, such as at the National Stem Cell Resource website, whichis maintained by the American Type Culture Collection (ATCC).

Fetus: A developing mammal at an embryonic stage before birth. Inhumans, the fetal stage of prenatal development starts at the beginningof the 9th week after fertilization. In human eyes and RPE are alreadyformed at 4 weeks from conception. RPE continues to mature for nextseveral weeks.

Fibroblast growth factor (FGF): Any suitable fibroblast growth factor,derived from any animal, and functional fragments thereof, such as thosethat bind the receptor and induce biological effects related toactivation of the receptor. A variety of FGFs are known and include, butare not limited to, FGF-1 (acidic fibroblast growth factor), FGF-2(basic fibroblast growth factor, bFGF), FGF-3 (int-2), FGF-4(hst/K-FGF), FGF-5, FGF-6, FGF-7, FGF-8, FGF-9 and FGF-98. “FGF” refersto a fibroblast growth factor protein such as FGF-1, FGF-2, FGF-4,FGF-6, FGF-8, FGF-9 or FGF-98, or a biologically active fragment ormutant thereof. The FGF can be from any animal species. In oneembodiment, the FGF is mammalian FGF, including but not limited to,rodent, avian, canine, bovine, porcine, equine and human. The amino acidsequences and method for making many of the FGFs are well known in theart.

The amino acid sequence of human bFGF and methods for its recombinantexpression are disclosed in U.S. Pat. No. 5,439,818, herein incorporatedby reference. The amino acid sequence of bovine bFGF and various methodsfor its recombinant expression are disclosed in U.S. Pat. No. 5,155,214,also herein incorporated by reference. When the 146 residue forms arecompared, their amino acid sequences are nearly identical, with only tworesidues that differ. Recombinant bFGF-2, and other FGFs, can bepurified to pharmaceutical quality (98% or greater purity) using thetechniques described in detail in U.S. Pat. No. 4,956,455.

An FGF inducer includes an active fragment of FGF. In its simplest form,the active fragment is made by the removal of the N-terminal methionine,using well-known techniques for N-terminal methionine removal, such as atreatment with a methionine aminopeptidase. A second desirabletruncation includes an FGF without its leader sequence. Those skilled inthe art recognize the leader sequence as the series of hydrophobicresidues at the N-terminus of a protein that facilitate its passagethrough a cell membrane but that are not necessary for activity and thatare not found on the mature protein. Human and murine bFGF arecommercially available.

Frizzled (Fzd): A family of seven-pass transmembrane mammalian proteinsthat have characteristics of G-protein-coupled receptors and that bindproteins of the Wnt family of lipoglycoproteins, secretedFrizzled-related proteins (sFRPs), R-spondin, and Norrin and activatesdownstream signaling. Frizzled proteins (also referred to as Frizzledreceptors) contain a cysteine-rich domain (CRD) that binds its cognateligands, a carboxy terminal PDZ (Psd-95/disc large/ZO-1homologous)-binding domain, and various consensus sites forserine/threonine kinases and tyrosine kinases Amino acid hydropathyanalysis indicates that the Frizzled proteins contain one extracellularamino terminus, three extracellular protein loops, three intracellularprotein loops, and an intracellular carboxy terminus.

Frizzled proteins have an important regulatory role during embryonicdevelopment and have also been associated, in humans and in animalmodels, with a number of diseases, including various cancers, cardiachypertrophy, familial exudative vitreoretinopathy, and schizophrenia.

There are at least 10 mammalian Frizzled proteins and the genes encodingthe mammalian Frizzled proteins are related to the Drosophila frizzledgenes. The human Frizzled proteins include Frizzled1 (Fzd1; GENBANK®Accession No. AB017363), Frizzled2 (Fzd2; GENBANK® Accession Nos.L37882/NM_001466), Frizzled3 (Fzd3; GENBANK® Accession No. AJ272427),Frizzled4 (Fzd4; GENBANK® Accession No. AB032417), Frizzled5 (Fzd5;GENBANK® Accession No. U43318), Frizzled6 (Fzd6; GENBANK® Accession No.AB012911), Frizzled7 (Fzd7; GENBANK® Accession No. AB010881), Frizzled8(Fzd8; GENBANK® Accession No. AB043703), Frizzled9 (Fzd9; GENBANK®Accession Nos. U82169/NM_003508) and Frizzled10 (Fzd10; GENBANK®Accession No. AB027464). All of the GENBANK® entries are incorporatedherein by reference as available on Jan. 1, 2013.

Growth factor: A substance that promotes cell growth, survival, and/ordifferentiation. Growth factors include molecules that function asgrowth stimulators (mitogens), factors that stimulate cell migration,factors that function as chemotactic agents or inhibit cell migration orinvasion of tumor cells, factors that modulate differentiated functionsof cells, factors involved in apoptosis, or factors that promotesurvival of cells without influencing growth and differentiation.Examples of growth factors are a fibroblast growth factor (such asFGF-2), epidermal growth factor (EGF), cilliary neurotrophic factor(CNTF), and nerve growth factor (NGF), and actvin-A.

Growth medium or expansion medium: A synthetic set of culture conditionswith the nutrients necessary to support the growth (cellproliferation/expansion) of a specific population of cells. In oneembodiment, the cells are stem cells, such as iPSCs. Growth mediagenerally include a carbon source, a nitrogen source and a buffer tomaintain pH. In one embodiment, growth medium contains a minimalessential media, such as DMEM, supplemented with various nutrients toenhance stem cell growth. Additionally, the minimal essential media maybe supplemented with additives such as horse, calf or fetal bovineserum.

Host cells: Cells in which a vector can be propagated and its DNAexpressed. The cell may be prokaryotic or eukaryotic. The term alsoincludes any progeny of the subject host cell. It is understood that allprogeny may not be identical to the parental cell since there may bemutations that occur during replication. However, such progeny areincluded when the term “host cell” is used.

Isolated: An “isolated” biological component, such as a nucleic acid,protein or organelle that has been substantially separated or purifiedaway from other biological components in the environment (such as acell) in which the component naturally occurs, i.e., chromosomal andextra-chromosomal DNA and RNA, proteins and organelles. Nucleic acidsand proteins that have been “isolated” include nucleic acids andproteins purified by standard purification methods. The term alsoembraces nucleic acids and proteins prepared by recombinant expressionin a host cell as well as chemically synthesized nucleic acids andproteins. Similarly, an “isolated” cell has been substantiallyseparated, produced apart from, or purified away from other cells of theorganism in which the cell naturally occurs. Isolated cells can be, forexample, at least 99%, at least 98%, at least 97%, at least 96%, 95%, atleast 94%, at least 93%, at least 92%, or at least 90% pure.

Mammal: This term includes both human and non-human mammals. Examples ofmammals include, but are not limited to: humans and veterinary andlaboratory animals, such as pigs, cows, goats, cats, dogs, rabbits andmice.

Marker or Label: An agent capable of detection, for example by ELISA,spectrophotometry, flow cytometry, immunohistochemistry,immunofluorescence, microscopy, Northern analysis or Southern analysis.For example, a marker can be attached to a nucleic acid molecule orprotein, thereby permitting detection of the nucleic acid molecule orprotein. Examples of markers include, but are not limited to,radioactive isotopes, nitroimidazoles, enzyme substrates, co-factors,ligands, chemiluminescent agents, fluorophores, haptens, enzymes, andcombinations thereof. Methods for labeling and guidance in the choice ofmarkers appropriate for various purposes are discussed for example inSambrook et al. (Molecular Cloning: A Laboratory Manual, Cold SpringHarbor, N.Y., 1989) and Ausubel et al. (In Current Protocols inMolecular Biology, John Wiley & Sons, New York, 1998).

In some embodiments, the marker is a fluorophore (“fluorescent label”).Fluorophores are chemical compounds, which when excited by exposure to aparticular wavelength of light, emits light (i.e., fluoresces), forexample at a different wavelength. Fluorophores can be described interms of their emission profile, or “color.” Green fluorophores, forexample Cy3, FITC, and Oregon Green, are characterized by their emissionat wavelengths generally in the range of 515-540λ. Red fluorophores, forexample Texas Red, Cy5 and tetramethylrhodamine, are characterized bytheir emission at wavelengths generally in the range of 590-690λ. Inother embodiments, the marker is a protein tag recognized by anantibody, for example a histidine (His)-tag, a hemagglutinin (HA)-tag,or a c-Myc-tag.

Membrane potential: The electrical potential of the interior of the cellwith respect to the environment, such as an external bath solution. Oneof skill in the art can readily assess the membrane potential of a cell,such as by using conventional whole cell techniques. The membranepotential can be assessed using many approaches, such as usingconventional whole cell access, or using, for example, perforated-patchwhole-cell and cell-attached configurations.

MicroRNA (miRNA): A single-stranded RNA molecule, which regulates geneexpression. miRNAs are encoded by genes from whose DNA they aretranscribed but miRNAs are not translated into protein; instead eachprimary transcript (a pri-miRNA) is processed into a short stem-loopstructure called a pre-miRNA and finally into a functional miRNA. MaturemiRNA molecules are either fully or partially complementary to one ormore messenger RNA (mRNA) molecules, and their main function is todown-regulate gene expression. MicroRNAs can be encoded by independentgenes, but also be processed (via the enzyme Dicer) from a variety ofdifferent RNA species, including introns, 3′ UTRs of mRNAs, longnoncoding RNAs, snoRNAs and transposons. As used herein, microRNAs alsoinclude “mimic” microRNAs which are intended to mean a microRNAexogenously introduced into a cell that have the same or substantiallythe same function as their endogenous counterpart.

Nodal: A secretory protein encoded by the NODAL gene that belongs to theTransforming Growth Factor (TGF-beta) superfamily During embryonicdevelopment, the left-right (LR) asymmetry of visceral organs invertebrates is established through nodal signaling. Nodal is expressedin the left side of the organism in early development and it is highlyconserved among deuterostomes. Exemplary Nodal sequences can be found asGENBANK® Accession Nos. NM_018055.4 and NP_060525.3, Jan. 6, 2013, whichare incorporated by reference herein.

Nucleic acid: A polymer composed of nucleotide units (ribonucleotides,deoxyribonucleotides, related naturally occurring structural variants,and synthetic non-naturally occurring analogs thereof) linked viaphosphodiester bonds, related naturally occurring structural variants,and synthetic non-naturally occurring analogs thereof. Thus, the termincludes nucleotide polymers in which the nucleotides and the linkagesbetween them include non-naturally occurring synthetic analogs, such as,for example and without limitation, phosphorothioates, phosphoramidates,methyl phosphonates, chiral-methyl phosphonates, 2-O-methylribonucleotides, peptide-nucleic acids (PNAs), and the like. Suchpolynucleotides can be synthesized, for example, using an automated DNAsynthesizer. It will be understood that when a nucleotide sequence isrepresented by a DNA sequence (i.e., A, T, G, C), this also includes anRNA sequence (i.e., A, U, G, C) in which “U” replaces “T.”

“Nucleotide” includes, but is not limited to, a monomer that includes abase linked to a sugar, such as a pyrimidine, purine or syntheticanalogs thereof, or a base linked to an amino acid, as in a peptidenucleic acid (PNA). A nucleotide is one monomer in a polynucleotide. Anucleotide sequence refers to the sequence of bases in a polynucleotide.

Conventional notation is used herein to describe nucleotide sequences:the left-hand end of a single-stranded nucleotide sequence is the5′-end; the left-hand direction of a double-stranded nucleotide sequenceis referred to as the 5′-direction. The direction of 5′ to 3′ additionof nucleotides to nascent RNA transcripts is referred to as thetranscription direction. The DNA strand having the same sequence as anmRNA is referred to as the “coding strand;” sequences on the DNA strandhaving the same sequence as an mRNA transcribed from that DNA and whichare located 5′ to the 5′-end of the RNA transcript are referred to as“upstream sequences;” sequences on the DNA strand having the samesequence as the RNA and which are 3′ to the 3′ end of the coding RNAtranscript are referred to as “downstream sequences.”

“cDNA” refers to a DNA that is complementary or identical to an mRNA, ineither single stranded or double stranded form.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(for example, rRNA, tRNA and mRNA) or a defined sequence of amino acidsand the biological properties resulting therefrom. Thus, a gene encodesa protein if transcription and translation of mRNA produced by that geneproduces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and non-codingstrand, used as the template for transcription, of a gene or cDNA can bereferred to as encoding the protein or other product of that gene orcDNA. Unless otherwise specified, a “nucleotide sequence encoding anamino acid sequence” includes all nucleotide sequences that aredegenerate versions of each other and that encode the same amino acidsequence. Nucleotide sequences that encode proteins and RNA may includeintrons. In some examples, a nucleic acid encodes a disclosed antigen.

Noggin: A protein which is encoded by the NOG gene. Noggin inhibitsTGF-β signal transduction by binding to TGF-β family ligands andpreventing them from binding to their corresponding receptors. Nogginplays a key role in neural induction by inhibiting BMP4, along withother TGF-β signaling inhibitors such as chordin and follistatin.Exemplary sequences for Noggin are GENBANK® Accession Nos. NP_005441.1and NM_005450.4, Jan. 13, 2013, which are incorporated herein byreference.

Oct-4: A protein also known as POU5-F1 or MGC22487 or OCT3 or OCT4 orOTF3 or OTF4, that is the gene product of the Oct-4 gene. The termincludes Oct-4 from any species or source and includes analogs andfragments or portions of Oct-4 that retain the ability to be used forthe production of iPSCs. The Oct-4 protein may have any of the knownpublished sequences for Oct-4 which can be obtained from public sourcessuch as GENBANK®. An example of such a sequence includes, but is notlimited to, GENBANK® Accession No. NM_002701.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein-coding regions, in the samereading frame.

Pharmaceutically acceptable carriers: Conventional pharmaceuticallyacceptable carriers are useful for practicing the methods and formingthe compositions disclosed herein. Remington's Pharmaceutical Sciences,by E. W. Martin, Mack Publishing Co., Easton, Pa., 15th Edition, 1975,describes examples of compositions and formulations suitable forpharmaceutical delivery of the antimicrobial compounds herein disclosed.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For example, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (e.g., powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate.

Pluripotent stem cells: Stem cells that: (a) are capable of inducingteratomas when transplanted in immunodeficient (SCID) mice; (b) arecapable of differentiating to cell types of all three germ layers (e.g.,can differentiate to ectodermal, mesodermal, and endodermal cell types);and (c) express one or more markers of embryonic stem cells (e.g.,express Oct 4, alkaline phosphatase, SSEA-3 surface antigen, SSEA-4surface antigen, nanog, TRA-1-60, TRA-1-81, SOX2, REX1, etc), but thatcannot form an embryo and the extraembryonic membranes (are nottotipotent).

Exemplary pluripotent stem cells include embryonic stem cells derivedfrom the inner cell mass (ICM) of blastocyst stage embryos, as well asembryonic stem cells derived from one or more blastomeres of a cleavagestage or morula stage embryo (optionally without destroying theremainder of the embryo). These embryonic stem cells can be generatedfrom embryonic material produced by fertilization or by asexual means,including somatic cell nuclear transfer (SCNT), parthenogenesis, andandrogenesis. PSCs alone cannot develop into a fetal or adult animalwhen transplanted in utero because they lack the potential to contributeto all extraembryonic tissue (e.g., placenta in vivo or trophoblast invitro).

Pluripotent stem cells also include “induced pluripotent stem cells(iPSCs)” generated by reprogramming a somatic cell by expressing orinducing expression of a combination of factors (herein referred to asreprogramming factors). iPSCs can be generated using fetal, postnatal,newborn, juvenile, or adult somatic cells. In certain embodiments,factors that can be used to reprogram somatic cells to pluripotent stemcells include, for example, Oct4 (sometimes referred to as Oct 3/4),Sox2, c-Myc, and Klf4, Nanog, and Lin28. In some embodiments, somaticcells are reprogrammed by expressing at least two reprogramming factors,at least three reprogramming factors, or four reprogramming factors toreprogram a somatic cell to a pluripotent stem cell. iPSCs are similarin properties to embryonic stem cells.

Polypeptide: A polymer in which the monomers are amino acid residuesthat are joined together through amide bonds. When the amino acids arealpha-amino acids, either the L-optical isomer or the D-optical isomercan be used, the L-isomers being preferred in nature. The termpolypeptide is specifically intended to cover naturally occurringproteins, as well as those that are recombinantly or syntheticallyproduced.

Substantially purified polypeptide as used herein refers to apolypeptide that is substantially free of other proteins, lipids,carbohydrates or other materials with which it is naturally associated.In one embodiment, the polypeptide is at least 50%, for example at least80% free of other proteins, lipids, carbohydrates or other materialswith which it is naturally associated. In another embodiment, thepolypeptide is at least 90% free of other proteins, lipids,carbohydrates or other materials with which it is naturally associated.In yet another embodiment, the polypeptide is at least 95% free of otherproteins, lipids, carbohydrates or other materials with which it isnaturally associated.

Conservative amino acid substitution tables providing functionallysimilar amino acids are well known to one of ordinary skill in the art.The following six groups are examples of amino acids that are consideredto be conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

A non-conservative amino acid substitution can result from changes in:(a) the structure of the amino acid backbone in the area of thesubstitution; (b) the charge or hydrophobicity of the amino acid; or (c)the bulk of an amino acid side chain. Substitutions generally expectedto produce the greatest changes in protein properties are those inwhich: (a) a hydrophilic residue is substituted for (or by) ahydrophobic residue; (b) a proline is substituted for (or by) any otherresidue; (c) a residue having a bulky side chain, e.g., phenylalanine,is substituted for (or by) one not having a side chain, e.g., glycine;or (d) a residue having an electropositive side chain, e.g., lysyl,arginyl, or histadyl, is substituted for (or by) an electronegativeresidue, e.g., glutamyl or aspartyl.

Variant amino acid sequences may, for example, be 80, 90 or even 95 or98% identical to the native amino acid sequence. Programs and algorithmsfor determining percentage identity can be found at the NCBI website.

Prenatal: Existing or occurring before birth. Similarly, “postnatal” isexisting or occurring after birth.

Primers: Short nucleic acid molecules, for instance DNA oligonucleotides10-100 nucleotides in length, such as about 15, 20, 25, 30 or 50nucleotides or more in length. Primers can be annealed to acomplementary target DNA strand (such as a gene listed in Table 1 orTable A, or miRNA listed in Table 2) by nucleic acid hybridization toform a hybrid between the primer and the target DNA strand. Primer pairscan be used for amplification of a nucleic acid sequence, such as by PCRor other nucleic acid amplification methods known in the art.

Methods for preparing and using nucleic acid primers are described, forexample, in Sambrook et al. (In Molecular Cloning: A Laboratory Manual,CSHL, New York, 1989), Ausubel et al. (ed.) (In Current Protocols inMolecular Biology, John Wiley & Sons, New York, 1998), and Innis et al.(PCR Protocols, A Guide to Methods and Applications, Academic Press,Inc., San Diego, Calif., 1990). PCR primer pairs can be derived from aknown sequence, for example, by using computer programs intended forthat purpose such as Primer (Version 0.5, © 1991, Whitehead Institutefor Biomedical Research, Cambridge, Mass.). One of ordinary skill in theart will appreciate that the specificity of a particular primerincreases with its length. Thus, for example, a primer including 30consecutive nucleotides of molecule will anneal to a target sequence,such as another homolog of the designated molecule, with a higherspecificity than a corresponding primer of only 15 nucleotides. Thus, inorder to obtain greater specificity, primers can be selected thatinclude at least 20, at least 25, at least 30, at least 35, at least 40,at least 45, at least 50 or more consecutive nucleotides of a nucleicacid sequence of interest.

Primer pairs: Two primers (one “forward” and one “reverse”) that can beused for amplification of a nucleic acid sequence, for example bypolymerase chain reaction (PCR) or other in vitro nucleic-acidamplification methods. The forward and reverse primers of a primer pairdo not hybridize to overlapping complementary sequences on the targetnucleic acid sequence.

Purified: The term “purified” does not require absolute purity; rather,it is intended as a relative term. Thus, for example, a purified proteinpreparation is one in which the protein referred to is more pure thanthe protein in its natural environment within a cell. For example, apreparation of a protein is purified such that the protein represents atleast 50% of the total protein content of the preparation. Similarly, apurified oligonucleotide preparation is one in which the oligonucleotideis more pure than in an environment including a complex mixture ofoligonucleotides. A purified population of cells is greater than about90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% pure, or freeother cell types.

Recombinant: A recombinant nucleic acid or polypeptide molecule is onethat has a sequence that is not naturally occurring or has a sequencethat is made by an artificial combination of two otherwise separatedsegments of sequence. This artificial combination can be accomplished bychemical synthesis of polypeptide or nucleic acid molecules, or by theartificial manipulation of isolated segments of nucleic acid molecules,such as by genetic engineering techniques.

Retinal pigment epithelial (RPE) cell: RPE cells can be recognized basedon pigmentation, epithelial morphology, and apical-basal polarizedcells. RPE cells express, both at the mRNA and protein level, one ormore of the following: Pax6, MITF, RPE65, CRALBP, PEDF, Bestrophinand/or Otx2. In certain other embodiments, the RPE cells express, bothat the mRNA and protein level, one or more of Pax-6, MitF, andtyrosinase. RPE cells do not express (at any detectable level) theembryonic stem cell markers Oct-4, nanog, or Rex-1. Specifically,expression of these genes is approximately 100-1000 fold lower in RPEcells than in ES cells or iPSC cells, when assessed by quantitativeRT-PCR. Differentiated RPE cells also can be visually recognized bytheir cobblestone morphology and the initial appearance of pigment. Inaddition, differentiated RPE cells have trans epithelial resistance/TER,and trans epithelial potential/TEP across the monolayer (TER>100ohms·cm²; TEP>2 mV), transport fluid and CO₂ from apical to basal side,and regulate a polarized secretion of cytokines.

The terms “RPE cell” and “differentiated RPE cell” and “iPSC-derived RPEcell” and “human RPE cell” are used interchangeably throughout to referto an RPE cell differentiated from a human iPSC using the methodsdisclosed herein. The term is used generically to refer todifferentiated RPE cells.

Retinal Pigment Epithelium: The pigmented layer of hexagonal cells,present in vivo in mammals, just outside of the neurosensory retinalthat is attached to the underlying choroid. These cells are denselypacked with pigment granules, and shield the retinal from incominglight. The retinal pigment epithelium also serves as the limitingtransport factor that maintains the retinal environment by supplyingsmall molecules such as amino acid, ascorbic acid and D-glucose whileremaining a tight barrier to choroidal blood borne substances.

Secreted Frizzled-related protein (sFRP): The sFRP family of proteinsare approximately 32-40 kDa glycoproteins that were identified asantagonists of Wnt signaling (Rattner et al. (1997) Proc. Natl. Acad.Sci. USA 94:2859-63; Melkonyan et al. (1997) Proc. Natl. Acad. Sci. USA94:13636-41; Finch et al. (1997) Proc. Natl. Acad. Sci. USA 94:6770-5;Uren et al. (2000) J. Biol. Chem. 275:4374-82; Kawano et al. (2003) J.Cell. Sci. 116:2627-34). In mammals, there are five sFRPs. The humansFRPs include sFRP1 (GENBANK® Accession No. AF001900.1), sFRP2 (GENBANK®Accession No. NM_003013.2), sFRP3 (GENBANK® Accession No. U91903.1),sFRP4 (GENBANK® Accession No. NM_003014.3), and sFRP5 (GENBANK®Accession No. NM_003015.3), as available on Jan. 1, 2013.

The sFRPs contain three structural units: an amino terminal signalpeptide, a Frizzled type cysteine-rich domain (CRD), and acarboxy-terminal netrin (NTR) domain. The CRD spans approximately 120amino acids, contains 10 conserved cysteine residues, and has 30-50%sequence similarity to the CRD of Fzd receptors. The netrin domain isdefined by six cysteine residues and several conserved segments ofhydrophobic residues and secondary structures. The biological activityof sFRPs is largely attributed to their role as regulators of Wntfunction.

Sequence identity: The similarity between two nucleic acid sequences orbetween two amino acid sequences is expressed in terms of the level ofsequence identity shared between the sequences. Sequence identity istypically expressed in terms of percentage identity; the higher thepercentage, the more similar the two sequences.

Methods for aligning sequences for comparison are well known in the art.Various programs and alignment algorithms are described in: Smith andWaterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J. Mol.Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. USA85:2444, 1988; Higgins and Sharp, Gene 73:237-244, 1988; Higgins andSharp, CABIOS 5:151-153, 1989; Corpet et al., Nucleic Acids Research16:10881-10890, 1988; Huang, et al., Computer Applications in theBiosciences 8:155-165, 1992; Pearson et al., Methods in MolecularBiology 24:307-331, 1994; Tatiana et al., (1999), FEMS Microbiol. Lett.,174:247-250, 1999. Altschul et al. present a detailed consideration ofsequence-alignment methods and homology calculations (J. Mol. Biol.215:403-410, 1990).

The National Center for Biotechnology Information (NCBI) Basic LocalAlignment Search Tool (BLAST™, Altschul et al. J. Mol. Biol.215:403-410, 1990) is available from several sources, including theNational Center for Biotechnology Information (NCBI, Bethesda, Md.) andon the Internet, for use in connection with the sequence-analysisprograms blastp, blastn, blastx, tblastn and tblastx. A description ofhow to determine sequence identity using this program is available onthe internet under the help section for BLAST™.

Sonic hedgehog (SHH): Sonic hedgehog (SHH) is one of three mammalianhomologs of the Drosophila hedgehog signaling molecule and is expressedat high levels in the notochord and floor plate of developing embryos.SHH is known to play a key role in neuronal tube patterning (Echerlardet al., Cell 75:1417-30, 1993), the development of limbs, somites, lungsand skin. Moreover, overexpression of SHH has been found in basal cellcarcinoma. Exemplary amino acid sequences of SHH is set forth in U.S.Pat. No. 6,277,820. An exemplary sequence for human Sonic is set forthas GENBANK Accession No. NG_007504.1 (Jan. 1, 2013), which isincorporated by reference herein.

Subject: An animal or human subjected to a treatment, observation orexperiment.

Totipotent or totipotency: A cell's ability to divide and ultimatelyproduce an entire organism including all extraembryonic tissues in vivo.In one aspect, the term “totipotent” refers to the ability of the cellto progress through a series of divisions into a blastocyst in vitro.The blastocyst comprises an inner cell mass (ICM) and a trophectoderm.The cells found in the ICM give rise to pluripotent stem cells (PSCs)that possess the ability to proliferate indefinitely, or if properlyinduced, differentiate in all cell types contributing to an organism.Trophectoderm cells generate extra-embryonic tissues, including placentaand amnion.

Treatment: Therapeutic measures that cure, slow down, lessen symptomsof, and/or halt progression of a diagnosed pathologic condition ordisorder. In certain embodiments, treating a subject with a retinaldisorder results in a decline in the deterioration of the retinal; anincrease in the number of retinal pigment epithelial cells, animprovement in vision, or some combination of effects.

Tyrosinase: A copper-containing oxidase that catalyzes the production ofmelanin and other pigments from tyrosine by oxidation. This enzyme isthe rate limiting enzyme for controlling the production of melanin.Tyrosinase acts in the hydroxylation of a monophenol and, the conversionof an o-diphenol to the corresponding o-quinone. o-Quinone undergoesseveral reactions to eventually form melanin. In humans, the tyrosinaseenzyme is encoded by the TYR gene. Exemplary amino acid and nucleic acidsequences are set forth in GENBANK® Accession Nos. NM_000372.4 (human)and NM_011661.4 (mouse), Jan. 5, 2013, and which are incorporated byreference herein.

Undifferentiated: Cells that display characteristic markers andmorphological characteristics of undifferentiated cells, distinguishingthem from differentiated cells of embryo or adult origin. Thus, in someembodiments, undifferentiated cells do not express cell lineage specificmarkers, including, but no limited to, RPE markers.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. A vector may include nucleic acidsequences that permit it to replicate in a host cell, such as an originof replication. A vector may also include one or more selectable markergenes and other genetic elements known in the art. A vector may alsoinclude a sequence encoding for an amino acid motif that facilitates theisolation of the desired protein product such as a sequence encodingmaltose binding protein, c-myc, or GST.

Wnt: A family of highly conserved secreted signaling molecules thatregulate cell-to-cell interactions and are related to the Drosophilasegment polarity gene, wingless. In humans, the Wnt family of genesencodes 38 to 43 kDa cysteine rich glycoproteins. The Wnt proteins havea hydrophobic signal sequence, a conserved asparagine-linkedoligosaccharide consensus sequence (see e.g., Shimizu et al Cell GrowthDiffer 8:1349-1358 (1997)) and 22 conserved cysteine residues. Becauseof their ability to promote stabilization of cytoplasmic beta-catenin,Wnt proteins can act as transcriptional activators and inhibitapoptosis. Overexpression of particular Wnt proteins has been shown tobe associated with certain cancers.

The Wnt family contains at least 19 mammalian members. Exemplary Wntproteins include Wnt-1, Wnt-2, Wnt2b, Wnt-3, Wnt-3a, Wnt-4, Wnt-5a,Wnt5b, Wnt-6, Wnt-7a, Wnt-7b, Wnt-8a, Wnt-8b, Wnt9a, Wnt9b, Wnt10a,Wnt-10b, Wnt-11, and Wnt 16. These secreted ligands activate at leastthree different signaling pathways. In the canonical (orWnt/beta-catenin) Wnt signaling pathway, Wnt activates a receptorcomplex consisting of a Frizzled (Fzd) receptor family member andlow-density lipoprotein (LDL) receptor-related protein 5 or 6 (LRP5/6).To form the receptor complex that binds the Fzd ligands, Fzd receptorsinteract with LRP5/6, single pass transmembrane proteins with fourextracellular EGF-like domains separated by six YWTD amino acid repeats(Johnson et al., 2004, J. Bone Mineral Res. 19:1749). The canonical Wntsignaling pathway activated upon receptor binding is mediated by thecytoplasmic protein Dishevelled (Dvl) interacting directly with the Fzdreceptor and results in the cytoplasmic stabilization and accumulationof beta-catenin. In the absence of a Wnt signal, beta-catenin islocalized to a cytoplasmic destruction complex that includes the tumorsuppressor proteins adenomatous polyposis coli (APC) and Axin. Theseproteins function as critical scaffolds to allow glycogen synthasekinase (GSK)-3beta to bind and phosphorylate beta-catenin, marking itfor degradation via the ubiquitin/proteasome pathway. Activation of Dvlresults in the dissociation of the destruction complex. Accumulatedcytoplasmic beta-catenin is then transported into the nucleus where itinteracts with the DNA-binding proteins of the TCF/LEF family toactivate transcription.

The non-canonical WNT pathway is regulated by three of these WNTligands—WNT4, WNT5a, and WNT11. These ligands bind to the WNT receptorFrizzled in the absence of the co-receptors (LRP5/6). This leads to theactivation of the RHO GTPase and ROCK kinase without activatingcytoplasmic beta-catenin. ROCK regulates cytoskeleton to regulateapical-basal polarity of the cell. Because of competition for the samereceptor, non-canonical WNT ligands also lead to inhibition of canonicalWNT signaling.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. It is further tobe understood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of this disclosure, suitable methods andmaterials are described below. The term “comprises” means “includes.”All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including explanations ofterms, will control. In addition, the materials, methods, and examplesare illustrative only and not intended to be limiting.

Methods for Reprogramming Somatic Cells to Produce RPE Cells

Methods are provided herein wherein induced pluripotent stem cells arereprogrammed to become retinal pigment epithelial cells. Stem cells canbe indefinitely maintained in vitro in an undifferentiated state and yetare capable of differentiating into virtually any cell type. Disclosedherein are methods to induce the differentiation of human iPSC into aspecialized cell in the neuronal lineage, the retinal pigment epithelium(RPE).

RPE is a densely pigmented epithelial monolayer between the choroid andneural retina that serves as a part of a barrier between the bloodstreamand retina. The functions of the RPE include phagocytosis of shed rodand cone outer segments, absorption of stray light, vitamin Ametabolism, regeneration of retinoids, and tissue repair. The RPE has acobblestone cellular morphology of black pigmented cells, and is knownto express markers such as cellular retinaldehyde-binding protein(CRALBP); RPE65, Best vitelliform macular dystrophy gene (VMD2), andpigment epithelium derived factor (PEDF). The RPE plays a role inphotoreceptor maintenance, and various RPE malfunctions in vivo areassociated with a number of vision-altering ailments, such as RPEdetachment, dysplasia, atrophy, retinopathy, retinitis pigmentosa,macular dystrophy or degeneration, including age-related maculardegeneration, which can result in photoreceptor damage and blindness.Because of its wound healing abilities, the RPE has been extensivelystudied for transplantation therapy; RPE transplantation can be used forvision restoration. Furthermore, RPE can be used for the treatment ofParkinson's disease, as the RPE secretes dopamine.

The starting somatic cell can be any cell of interest. Any cells otherthan germ cells of mammalian origin (such as, humans, mice, monkeys,pigs, rats etc.) can be used as starting material for the production ofiPSCs. Examples include keratinizing epithelial cells, mucosalepithelial cells, exocrine gland epithelial cells, endocrine cells,liver cells, epithelial cells, endothelial cells, fibroblasts, musclecells, cells of the blood and the immune system, cells of the nervoussystem including nerve cells and glia cells, pigment cells, andprogenitor cells, including hematopoietic stem cells, amongst others.There is no limitation on the degree of cell differentiation, the age ofan animal from which cells are collected and the like; evenundifferentiated progenitor cells (including somatic stem cells) andfinally differentiated mature cells can be used alike as sources ofsomatic cells in the present invention. In one embodiment, the somaticcell is itself a RPE cell such as a human RPE cell. The RPE cell can bean adult or a fetal RPE cell. Thus, in some embodiments, the somaticcell is a fetal human RPE cell, obtained from a human fetus of aboutweek 9 to about week 38 of gestation, such as about week 9 to about week16 of gestation, about week 17 to about week 25 of gestation, about 16to about 19 weeks, or about week 26 to about week 38 of gestation. Inthis context, “about” means within 2 days.

The choice of mammalian individuals as a source of somatic cells is notparticularly limited. Allogenic cells can be used. Thus, in someembodiments, the iPSCs are not matched for MHC (e.g., HLA). In someembodiments, when the iPSCs obtained are to be used for regenerativemedicine in humans, cells can be collected from the somatic cells fromthe subject to be treated, or another subject with the same orsubstantially the same HLA type as that of the patient. “Substantiallythe same HLA type” indicates that the HLA type of donor matches withthat of a patient to the extent that the transplanted cells, which havebeen obtained by inducing differentiation of iPSCs derived from thedonor's somatic cells, can be engrafted when they are transplanted tothe patient. The patient optionally can be treated with animmunosuppressant. In one example, it includes an HLA type wherein majorHLAs (e.g., the three major loci of HLA-A, HLA-B and HLA-DR, the fourmajor loci further including HLA-Cw) are identical.

Somatic cells isolated from a mammal can be pre-cultured using a mediumknown to be suitable for their cultivation according to the choice ofcells before being subjected to the step of nuclear reprogrammingSpecific non-limiting examples of such media include, but are notlimited to, minimal essential medium (MEM) containing about 5 to 20%fetal calf serum (FCS), Dulbecco's modified Eagle medium (DMEM),RPMI1640 medium, 199 medium, F12 medium, and the like. One of skill inthe art can readily ascertain appropriate tissue culture conditions topropagate particular cell types from a mammal, such as a human. In someembodiments, to obtain completely xeno-free human iPSCs, the medium canexclude ingredients derived from non-human animals, such as FCS. Mediacomprising a basal medium supplemented with human-derived ingredientssuitable for cultivation of various somatic cells (particularly,recombinant human proteins such as growth factors), non-essential aminoacids, vitamins and the like are commercially available; those skilledin the art are able to choose an appropriate xeno-free medium accordingto the source of somatic cells. Somatic cells pre-cultured using axeno-free medium are dissociated from the culture vessel using anappropriate xeno-free cell dissociation solution, and recovered, afterwhich they are brought into contact with nuclear reprogrammingsubstances.

Generally, cells are cultured at about 35 to 38° C., usually at 37° C.,in about 4-6% CO₂, generally at 5% CO₂, unless specifically indicatedotherwise below.

Somatic cells can be reprogrammed to produce induced pluripotent stemcells (iPSCs) using methods known to one of skill in the art. One ofskill in the art can readily produce induced pluripotent stem cells, seefor example, Published U.S. Patent Application No. 20090246875,Published U.S. Patent Application No. 2010/0210014; Published U.S.Patent Application No. 20120276636; U.S. Pat. Nos. 8,058,065; 8,129,187;8,278,620; PCT Publication NO. WO 2007/069666 A1, and U.S. Pat. No.8,268,620, which are incorporated herein by reference. Generally,nuclear reprogramming factors are used to produce pluripotent stem cellsfrom a somatic cell. In some embodiments, at least three, or at leastfour, of Klf4, c-Myc, Oct3/4, Sox2, Nanog, and Lin28 are utilized. Inother embodiments, Oct3/4, Sox2, c-Myc and Klf4 is utilized.

The cells are treated with a nuclear reprogramming substance, which isgenerally one or more factor(s) capable of inducing an iPSC from asomatic cell or a nucleic acid that encodes these substances (includingforms integrated in a vector). The nuclear reprogramming substancesgenerally include at least Oct3/4, Klf4 and Sox2 or nucleic acids thatencode these molecules. A functional inhibitor of p53, L-myc or anucleic acid that encodes L-myc, and Lin28 or Lin28b or a nucleic acidthat encodes Lin28 or Lin28b, can be utilized as additional nuclearreprogramming substances. Nanog can also be utilized for nuclearreprogramming. As disclosed in published U.S. Patent Application No.20120196360, exemplary reprogramming factors for the production of iPSCsinclude (1) Oct3/4, Klf4, Sox2, L-Myc (Sox2 can be replaced with Sox1,Sox3, Sox15, Sox17 or Sox18; Klf4 is replaceable with Klf1, Klf2 orKlf5); (2) Oct3/4, Klf4, Sox2, L-Myc, TERT, SV40 Large T antigen(SV40LT); (3) Oct3/4, Klf4, Sox2, L-Myc, TERT, human papilloma virus(HPV)16 E6; (4) Oct3/4, Klf4, Sox2, L-Myc, TERT, HPV16 E7 (5) Oct3/4,Klf4, Sox2, L-Myc, TERT, HPV16 E6, HPV16 E7; (6) Oct3/4, Klf4, Sox2,L-Myc, TERT, Bmi1; (7) Oct3/4, Klf4, Sox2, L-Myc, Lin28; (8) Oct3/4,Klf4, Sox2, L-Myc, Lin28, SV40LT; (9) Oct3/4, Klf4, Sox2, L-Myc, Lin28,TERT, SV40LT; (10) Oct3/4, Klf4, Sox2, L-Myc, SV40LT; (11) Oct3/4,Esrrb, Sox2, L-Myc (Esrrb is replaceable with Esrrg); (12) Oct3/4, Klf4,Sox2; (13) Oct3/4, Klf4, Sox2, TERT, SV40LT; (14) Oct3/4, Klf4, Sox2,TERT, HPV16 E6; (15) Oct3/4, Klf4, Sox2, TERT, HPV16 E7; (16) Oct3/4,Klf4, Sox2, TERT, HPV16 E6, HPV16 E7; (17) Oct3/4, Klf4, Sox2, TERT,Bmi1; (18) Oct3/4, Klf4, Sox2, Lin28 (19) Oct3/4, Klf4, Sox2, Lin28,SV40LT; (20) Oct3/4, Klf4, Sox2, Lin28, TERT, SV40LT; (21) Oct3/4, Klf4,Sox2, SV40LT; or (22) Oct3/4, Esrrb, Sox2 (Esrrb is replaceable withEsrrg). In one non-limiting example, Oct3/4, Klf4, Sox2, and c-Myc areutilized. In other embodiments, Oct4, Nanog, and Sox2 are utilized, seefor example, U.S. Pat. No. 7,682,828, which is incorporated herein byreference. These factors include, but are not limited to, Oct3/4, Klf4and Sox2. In other examples, the factors include, but are not limited toOct 3/4, Klf4 and Myc. In some non-limiting examples, Oct3/4, Klf4,c-Myc, and Sox2 are utilized. In other non-limiting examples, Oct3/4,Klf4, Sox2 and Sal 4 are utilized.

Mouse and human cDNA sequences of these nuclear reprogramming substancesare available with reference to the NCBI accession numbers mentioned inWO 2007/069666, which is incorporated herein by reference. Methods forintroducing one or more reprogramming substances, or nucleic acidsencoding these reprogramming substances, are known in the art, anddisclosed for example, in published U.S. Patent Application No.2012/0196360 and U.S. Pat. No. 8,071,369, which both are incorporatedherein by reference.

After being cultured with nuclear reprogramming substances, the cellcan, for example, be cultured under conditions suitable for culturing EScells. In the case of mouse cells, the culture is carried out with theaddition of Leukemia Inhibitory Factor (LIF) as a differentiationsuppression factor to an ordinary medium. In the case of human cells, itis desirable that basic fibroblast growth factor (bFGF) be added inplace of LIF.

In some embodiments, the cell is cultured in the co-presence of mouseembryonic fibroblasts treated with radiation or an antibiotic toterminate the cell division, as feeder cells. Mouse embryonicfibroblasts in common use as feeders include the STO cell line (ATCCCRL-1503) and the like; for induction of an iPSC, useful cells can begenerated by stably integrating the neomycin resistance gene and the LIFgene in the STO cell (SNL76/7 STO cell; ECACC 07032801) (McMahon, A. P.& Bradley, A. Cell 62, 1073-1085, 1990) and the like can be used.Mitomycin C-treated MEFs are commercially available from Millipore.Gamma-irradiated MEFs are commercially available from Global StemGenerally, somatic cells are transduced with reprogramming factors inthe absence of MEFs. In some embodiments, about 7 to eight days aftertransduction, the cells are re-seeded onto MEFs.

In some embodiments, the iPSC can be modified to express exogenousnucleic acids, such as to include a tyrosinase enhancer operably linkedto a promoter and a nucleic acid sequence encoding a first marker. Thetyrosinase gene is disclosed, for example, in GENBANK® Accession No.22173, as available on Jan. 1, 2013. This sequence aligns to chromosome7 of mouse strain C57BL/6 location 5286971-5291691 (invert orientation).A 4721 base pair sequence is sufficient for expression in RPE cells, seeMurisier et al., Dev. Biol. 303: 838-847, 2007, which is incorporatedherein by reference. This construct is expressed in retinal pigmentepithelial cells. In some embodiments, the tyrosinase enhancer sequencecomprises or consists of the following nucleic acid sequence:

(SEQ ID NO: 1) TTCTTTTGCCCTTTCCTTTTCATAAACTGAACTTCATTTTAAGCAACAAGTCTGTGTGAAACAGAAATGTCCTAATCTCTCTTTGACCAAATGTACCCATATTCCCTTATGTTAACATGTATTTTTTACATTTAAGATTGTTAAAGTGGAACAGTTTTTTTTCTGCCATTATAGCACCTGTCTCTACTTTTCAAAGTATATGAATTATGATCTTTCTCATGTGGTTGTAAGCCCCATCTTTACAAGATTCACTTGATCTTTCATATTCAATTATTTATGGAACAAAATACCTGTCAATTCTTAGAGTCTTTTCTACATAATTTATTTGTGAAAGAAAATGTTACTGGAAAGTGACAAATTAGAGTCAAAATATAAAGACTGTGGCAGGTTATATACCTATAGTGTGATATGAAAGCTTTTGTAAGAAGAGGTAGTGGTACTAAACTGGACAAAATCCAGATAAAAGAGGCTTTGTGAAAATCAGGTAAAAATTTACTTAATATACAGCAAATACTAATAGTTGCTGTTTATAAAATACCATTTTCTGAACATTGTTTTTGCACATATAACTAAAATGTTGAATATACCCAAGTATGAAAATTTAGTGTCATAGATATTAAGAACATTCTACCCTTTTCAGGAGAGTCATTGACAGTGATTTAAGTGACTCTGCTTACACTGCTTGTCCTCTAACACTGACTCCATAATGATTGCAGCAAAAAATTAAAGCTCAAACGGTCTTGGGGATTACCTAGTTCAATGACTTTGATTTAACACAGTAAGTACTTGAAGTAGAAAGAGGTACATTAACAAGCCAGGCAATGATGATATGGAGGGCAGTGTGATTAGAGTACAGGATTCTACACTCATTCTGCATTATTGGTTCATATTTCTTCTGGGGTAACTCACTTCCTTCCTTTTTCATATTTTCATTGCTCTAACTCTAGCCTTGACTTTAGGAACATCTCTTCTTTTTCCACCTATAAGATAGAATTGTTTTCTGCTGCAGGAGATTAAGATAGCTGGCATTCCTTTATGCTTATTTAGTCATTTCAAGCGATTAACTTCATCCTATCAGACTTTGAGATTAAGCTGCCAGTAGTGACCTCATTAAAGTCCACACTTCTAATAAGCTTCTCTAAAAATTGTTGAGAAGGCATTCTTGAGTTGGTACAGGGAAAGAATTGTGGACTCAGAAGCAAACATAGCAAAGCTCATTTGTTCCAGTCTATGGTTTACAGGTCAAGTGATTTGGGACCAATTGCCAAAATACATTGGTGAGGAAAGGCATTAATATCAACTATGCCAAGTTACTATGCTTATTAAACTCAACCATGATACAGAGTTATATAATGTTATAATGTATTCATTGAATGTTTTATAAGAAACCAATTGTTTATTTGTTATTTAACTCTGCAAAACTACAGAAAGGGGAAATGGTTATTTAAGTGGGTAATAAGTTTTAAGTATTTATCGTTCATAATAATTAACAGAGATGTTACAAAAATGTGACTGATTTTACTTGAAATGTTGCCATTTTAGTAAATGTGGTGCCAAAGCAAGCATGAAATGTTGCCATTTTAAAGACATTTATTTTCTAATGCTATAATATATTCATTACATATTATTAAAATAATTAATGTAAAAATACCCAAAATGTGAAAATAACACGTAAGTCCTATTTTATGATTTTCCATATCAAATTCAGAAACTAATACTCAGATCTTATTGTTTAAATAGTATTTAAAATTAAAGACACATAGTCAGGAATATATGCTAAATAAATTTTCCAAATTGAATAACTAACTTTCAGGGTTGCCTTACTTTCAACAAGAATATGCCTCTATTTGATTACTAATTGTAACTTTGTTCATAGACTACATAAGGTAATATTACAAACATATTCATTATTTTGACACATACTTACTTAAATAATAAATAAACATTAGAAAATATACTTACTATTTATATATAAAGAATTTTTTTGTTTCGAAGGAACTTTAATAAATGAGATTATAAGGTTGTTGTTCAGGTACATTGAACATTTTTTCAGGTTAATAAGGTGTCTAAAAATAAGTTTAGAAAGATCTAAGGTATTCTTTTTTATTTGTTTTTGTCCTTTTTTATTTTTCTTTTTTGAACTGGGTTTCTTCTTCAATTAGCCCTGGCTGGATGTCCTGGAACTAGCTCTATATGCCATGCTGTCTTCAAACTCATAGAGATCTGCCTACCTTTGCATCCCAAGTGCTGGGATTAAAGGTATGAACCACCTCTTCCACCACTGCCAAGTAAGAATTCTTATTTTACATAAGTCATTATGAAGGAAGTTATGTGTTTACTGTAAACAAGATTAATGACTTGGTTTGCTGATTTTCTCTGAAAACATGAAATCTCTTCATATAGATCTTGCTTCTGGATAATAAAAGGCCCATGGAGAAATGTTCGTCTGTCTAGTTTCATATTCATATTAATGATCCTGAATCAATTTTCCTCCATTGAGACTTGCATACTTAAGTATTAATGATTGCTGGAGTTCCATTCATAAGGATTCTTTATGTATTACATGTTAAAATTTTTAAACCTACGACATTTTGGGATATAGTTTAGTAAAACATCTTAAATGGTGTAAGTGGTACCAATTAGTTTGAAGGCAAAAACAATTGTTTAAGTGGATTAAGGTCTGTTCAATACTAGGGAACACCTGCTTGAAACACTTGACAATAGAAACTTAGCTAACTTACCCATGTCTGGAAAGGTCATGGACTCTGAAGGAAAACTACTTTTACCATTTTCCTAAATCAATATAGCTTTTAACTATTCTAAACACTGATCATTATACCCATAGACAGTTTAGATCAACCCTTTCCAAAGAAGATTCTGTTTGTAGTAGATAAGGCTTAATACAAAGATCCTCAATTGGTCCAAATCCAGAGAATAGGTAAGCCTGTGGTGTTTAGGTGCCCAACTTAGTCTACCAATAATACTACCTATGTACTTGAGTACTAGTGAACAGGATACAGAAGGTGGTATCAGGAAGACTGTAAGAACCAGAAAATATGAATACATACATGTGTATATTTATGCAAGAGTAACAATTAAAGAAGTTTATTAATCTGAGTGTGTATGTGTTAATGTATAAAAATCTCAGAAAAGTATTTAAAATTATTTTGCCTTTGGAAATAAAAATAACAAGTATTGTTCAAGAAAAAGATAATTCCAGGATACTAGCCAATTTTGCTCTTAACTTAGAAATATAATTATATTTTTTCTTCTCTTTGACTGGATAAATATGTACGAATGTTCTTTGAATATTTGCAGCCAATTTGACTCCCTAAAAAATGGTATAGTTTTAAATGTGTTTAACATATTGCTTTTGTGAAAGACATTTTTTTAGTATTAGATTCAATACTTTTTAACCATGTGGACATGGTTGGTGTTATTTTTGTTCTAGAAAGGAACTGTTAAATTTCTGCTCCAACTTAGGTCATATAAGGGAAAATGAATCTGGTATTCTACAGAAAAATATAACTGTAACCATTTTGATGATTTTTGTGTTAATTAGCACTGTTTGTCTGTTCATATCATTGAGGCACAGAAATGGTATATTTATATAACACCTACCAAACAGCCTCATAAAGAAATAGATAGATTCTGGGGAATAAATGATCTCCATTTGATCCTCAGTTTTATTAAAATCCTTCTGTTCCTGTGGCATGAATTCATCCAAACTGAGTAATGCTGGCAAGCAGGAAGGGGATCAAGGTCATCCAAGGGATACTGACTTGGAAGGGTCTGGGCATGCAACCAAGTACTTCCAGGGTGAATTATTATTAAGAAAAAGAATGTTGACAAAAAAATATGTGAAAAGGACCTATAGCTAGCTATTCTCTTGGTGACCTGGGTCTTGAGGAAGTTCTCTGGGAAGAGTCACTCAGCACATTTGGTCAAATGAATTCACCTATTCTGAAAACCAAATGAATATAGATTTCTGGACACCTCCCAAGGATTCATGTGTAAGATGAAATGCAGATTGTTCACCAAAATTGTCCCTGACTCCTATACTTAGACCATTTATTTTTCTGAAATCCCATAAACTGAGAAGATGCTGTCTGATTAGAAGATACACAAGTCGTGGATAATAAGACAAAAGAGCCCATGAACCTACAAAGCTCATTGCAAAGTGAACTTCTGTCTTGTAACAGAGAAAGCAGACAAACCAACAAAATCATTTATTTCAGTGAAAAGGAGGGGCCAGAAATGGAAAGATTACATTTCCTAAGTCTCGTACTTGAAGACAGGTTGGGTCCTCAGAACTAATTAAGTAGTAGAATGCACAATGTGCTTCAAGAAAAAAGAAGCTATGAAAAACAGGTAGTCTATTTTATTTCAACCTAGCAACAGTGAGAAAAGGATGAGCTAGCAAGGAGATGCAGATAGTGAAGTGTCCATTGTGGATTTACTCTGGTTCTGACAGGTGGAATTGCTTCCATTCAAAACAAACAAAATAAACTTCTAACTCACAGTAATTCACAGTGTCACACTTTGTAACACAGGATGTCAAAGTTTCAGGACATACAGTCTCAACACATAGGTAATTAATTTAAGTGAGGTGATTTGAGTGAATTTAAATGCAATGGACTTGTAGATTTTGTAAAAAGAAGACACGTCTTTCAATACGCACACATATGGGAAAATGGTAT GTAAATATGAAGTTAGCACTT

Longer fragments of the upstream region of the tyrosinase gene, thatinclude this enhancer, can also be utilized, such as 5 kb, 6 Kb, 7 kb, 8kb, etc.

Any promoter can be utilized, including, but not limited to Hsp70 1apromoter (GENBANK® Accession No. NT_039649.8. In some embodiment, thepromoter includes, or consists of:

(SEQ ID NO: 2) CAGGAACATCCAAACTGAGCAGCCGGGGTCCCCCCCACCCCCCACCCCGCCCCTCCCGGCAACTTTGAGCCTGTGCTGGGACAGAGCCTCTAGTTCCTAAATTAGTCCATGAGGTCAGAGGCAGCACTGCCATTGTAACGCGATTGGAGAGGATCACGTCACCGGACACGCCCCCAGGCATCTCCCTGGGTCTCCTAAACTTGGCGGGGAGAAGTTTTAGCCCTTAAGTTTTAGCCTTTAACCCCCATATTCAGAACTGTGCGAGTTGGCGAAACCCCACAAATCACAACAAACTGTACACAACACCGAGCTAGAGGTGATCTTTCTTGTCCATTCCACACAGGCCTTAGTAATGCGTCGCCATAGCAACAGTGTCACTAGTAGCACCAGCACTTCCCCACACCCTCCCCCTCAGGAATCCGTACTCTCCAGTGAACCCCAGAAACCTCTGGAGAGTTCTGGACAAGGGCGGAACCCACAACTCCGATTACTCAAGGGAGGCGGGGAAGCTCCACCAGACGCGAAACTGCTGGAAGATTCCTGGCCCCAAGGCCTCCTCCGGCTCGCTGATTGGCCCAGCGGAGAGTGGGCGGGGCCGGTGAAGACTCCTTAAAGGCGCAGGGCGGCGAGCAGGTCACCAGACGCTGACAGCTACTCAGAACCAAATCTGGTTCCATCCAGAGACAAGCGAAAGACAAGAGAAGCAGAGCGAGCGGCGCGTTCCCGATCCTCGGCCAGGACCAGCCTTCCCCAGAGCATCCCTGCCGCGGAGCGCAACCTTCCCAGGAGCATCCCTGCCGCGGAGCGCAACTTTCCCCGGAGCATCCACGGCCGCGGAGCGCAGCCTTTCCAGAAGCAGAAGCGCGGCGCCAATGGCTCGCGAATGAATCCCGTCGGTTTTAACAAACGGTCGGTGAACCTGGGGAAAAACCTGCGGTTAACCCAACTTAAATTCGCCCTCTGGGCAAGAC 

Small deletions, additions, and substitutions can be made withoutaffecting the activity of SEQ ID NO: 1 and/or SEQ ID NO: 2, such as atmost 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 base.

Other enhancers can be utilized. Other RPE-specific enhancers includeD-MITF, DCT, TYRP1, RPE65, VMD2, MERTK, MYRIP, RAB27A, Suitablepromoters include, but are not limited to, any promoter expressed inretinal pigment epithelial cells including the tyrosinase promoter. Theconstruct can also include other elements, such as a ribosome bindingsite for translational initiation (internal ribosomal bindingsequences), and a transcription/translation terminator.

Generally, it is advantageous to transfect cells with the construct.Suitable vectors for stable transfection include, but are not limited toretroviral vectors, lentiviral vectors and Sendai virus.

Plasmids have been designed with a number of goals in mind, such asachieving regulated high copy number and avoiding potential causes ofplasmid instability in bacteria, and providing means for plasmidselection that are compatible with use in mammalian cells, includinghuman cells. Particular attention has been paid to the dual requirementsof plasmids for use in human cells. First, they are suitable formaintenance and fermentation in E. coli, so that large amounts of DNAcan be produced and purified. Second, they are safe and suitable for usein human patients and animals. The first requirement calls for high copynumber plasmids that can be selected for and stably maintainedrelatively easily during bacterial fermentation. The second requirementcalls for attention to elements such as selectable markers and othercoding sequences. In some embodiments plasmids that encode a marker arecomposed of: (1) a high copy number replication origin, (2) a selectablemarker, such as, but not limited to, the neo gene for antibioticselection with kanamycin, (3) transcription termination sequences,including the tyrosinase enhancer and (4) a multicloning site forincorporation of various nucleic acid cassettes; and (5) a nucleic acidsequence encoding a marker operably linked to the tyrosinase promoter.There are numerous plasmid vectors that are known in the art forinducing a nucleic acid encoding a protein. These include, but are notlimited to, the vectors disclosed in U.S. Pat. Nos. 6,103,470;7,598,364; 7,989,425; and 6,416,998, which are incorporated herein byreference.

Viral vectors can be utilized for the introduction of nucleic acids,including polyoma, SV40 (Madzak et al., 1992, J. Gen. Virol.,73:15331536), adenovirus (Berkner, 1992, Cur. Top. Microbiol. Immunol.,158:39-6; Berliner et al., 1988, Bio Techniques, 6:616-629; Gorziglia etal., 1992, J. Virol., 66:4407-4412; Quantin et al., 1992, Proc. Nacl.Acad. Sci. USA, 89:2581-2584; Rosenfeld et al., 1992, Cell, 68:143-155;Wilkinson et al., 1992, Nucl. Acids Res., 20:2233-2239;Stratford-Perricaudet et al., 1990, Hum. Gene Ther., 1:241-256),vaccinia virus (Mackett et al., 1992, Biotechnology, 24:495-499),adeno-associated virus (Muzyczka, 1992, Curr. Top. Microbiol. Immunol.,158:91-123; On et al., 1990, Gene, 89:279-282), herpes viruses includingHSV and EBV (Margolskee, 1992, Curr. Top. Microbiol. Immunol.,158:67-90; Johnson et al., 1992, J. Virol., 66:29522965; Fink et al.,1992, Hum. Gene Ther. 3:11-19; Breakfield et al., 1987, Mol. Neurobiol.,1:337-371; Fresse et al., 1990, Biochem. Pharmacol., 40:2189-2199),Sindbis viruses (H. Herweijer et al., 1995, Human Gene Therapy6:1161-1167; U.S. Pat. Nos. 5,091,309 and 5,2217,879), alphaviruses (S.Schlesinger, 1993, Trends Biotechnol. 11:18-22; I. Frolov et al., 1996,Proc. Natl. Acad. Sci. USA 93:11371-11377), human herpesvirus vectors(HHV) such as HHV-6 and HHV-7, and retroviruses of avian (Brandyopadhyayet al., 1984, Mol. Cell Biol., 4:749-754; Petropouplos et al., 1992, J.Virol., 66:3391-3397), murine (Miller, 1992, Curr. Top. Microbiol.Immunol., 158:1-24; Miller et al., 1985, Mol. Cell Biol., 5:431-437;Sorge et al., 1984, Mol. Cell Biol., 4:1730-1737; Mann et al., 1985, J.Virol., 54:401-407), and human origin (Page et al., 1990, J. Virol.,64:5370-5276; Buchschalcher et al., 1992, J. Virol., 66:2731-2739).Baculovirus (Autographa californica multinuclear polyhedrosis virus;AcMNPV) vectors can be used. Vectors can be obtained from commercialsources (such as PharMingen, San Diego, Calif.; Protein Sciences Corp.,Meriden, Conn.; Stratagene, La Jolla, Calif.). Suitable vectors aredisclosed, for example, in U.S. Published Patent Application No.2010/0247486, which is incorporated herein by reference. In specificnon-limiting examples, the vectors are retrovirus vectors (for example,lentivirus vectors), measles virus vectors, alphavirus vectors,baculovirus vectors, Sindbis virus vectors, adenovirus and poliovirusvectors.

Methods of transfection of DNA as calcium phosphate coprecipitates,conventional mechanical procedures such as microinjection,electroporation, insertion of a plasmid encased in liposomes, or virusvectors may be used. Eukaryotic cells can also be cotransformed withpolynucleotide sequences encoding the antibody, labeled antibody, orfunctional fragment thereof, and a second foreign DNA molecule encodinga selectable phenotype, such as the herpes simplex thymidine kinasegene.

A viral gene delivery system can be an RNA-based or DNA-based viralvector. An episomal gene delivery system can be a plasmid, anEpstein-Barr virus (EBV)-based episomal vector, a yeast-based vector, anadenovirus-based vector, a simian virus 40 (SV40)-based episomal vector,a bovine papilloma virus (BPV)-based vector, or a lentiviral vector.

Markers include, but are not limited to, fluorescence proteins (forexample, green fluorescent protein or red fluorescent protein), enzymes(for example, horse radish peroxidase or alkaline phosphatase orfirefly/renilla luciferase or nanoluc), or other proteins. A marker maybe a protein (including secreted, cell surface, or internal proteins;either synthesized or taken up by the cell); a nucleic acid (such as anmRNA, or enzymatically active nucleic acid molecule) or apolysaccharide. Included are determinants of any such cell componentsthat are detectable by antibody, lectin, probe or nucleic acidamplification reaction that are specific for the marker of the cell typeof interest. The markers can also be identified by a biochemical orenzyme assay or biological response that depends on the function of thegene product. Nucleic acid sequences encoding these markers can beoperably linked to the tyrosinase enhancer. In addition, other genes canbe included, such as genes that may influence stem cell to RPEdifferentiation, or RPE function, or physiology, or pathology. Thus, insome embodiments, a nucleic acid is included that encodes one or more ofMITF, PAX6, TFEC, OTX2, LHX2, VMD2, CFTR, RPE65, MFRP, CTRP5, CFH, C3,C2B, APOE, APOB, mTOR, FOXO, AMPK, SIRT1-6, HTRP1, ABCA4, TIMP3, VEGFA,CFI, TLR3, TLR4, APP, CD46, BACE1, ELOLV4, ADAM10, CD55, CD59, andARMS2.

The iPSCs, optionally including the tyrosinase promoter operably linkedto a marker, are cultured under conditions such that embryoid bodies(EBs) are formed. Methods for the production of EBs are known in theart. In some embodiments, EBs are produced in suspension culture:undifferentiated iPSCs are harvested by brief collagenase digestion,dissociated into clusters, and cultured in non-adherent cell cultureplates EBs are cultured for about 36 to about 72 hours, such as forabout 48 hours and then plated. In some examples, the medium is notchanged during this period.

Without being bound by theory, EBs are formed by the homophilic bindingof the Ca2+ dependent adhesion molecule E-cadherin, which is highlyexpressed on undifferentiated stem cells. When cultured as single cellsunder specific conditions, iPSCs spontaneously aggregate to form EBs.Such spontaneous formation is often accomplished in bulk suspensioncultures whereby the dish is coated with non-adhesive materials, such asagar or hydrophilic polymers, to promote the preferential adhesionbetween single cells, rather than to the culture substrate. To avoiddissociation into single cells, EBs can be formed from iPSCs by manualseparation of adherent colonies (or regions of colonies) andsubsequently cultured in suspension. Formation of EBs in suspension isamenable to the formation of large quantities of EBs, but provideslittle control over the size of the resulting aggregates, often leadingto large, irregularly shaped EBs. As an alternative, the hydrodynamicforces imparted in mixed culture platforms increase the homogeneity ofEB sizes when iPSCs are inoculated within bulk suspensions.

In some embodiments, EBs are selected that include about 150 to about600 cells, such as about 200 to about 500 cells, such as about 200 toabout 500 cells. In additional embodiments, the EBs include less thanabout 500 cells, such as less than about 450 cells or less than about400 cells. In further embodiments, the EBs include less than 500 cells,such as less than 450 cells or less than 400 cells. In otherembodiments, about 200 to about 400 EBs are plated in each well of astandard 6-well tissue culture plate, such as about 100 to about 200EBs, for example about 100 to about 150 EBs. In this context, “about”means within 20 cells or embryoid bodies.

The EBs are then cultured a first medium comprising two Wnt pathwayinhibitors and a Nodal pathway inhibitor. The first medium, thatincludes the Wnt pathway inhibitors and the Nodal pathway inhibitor, canbe a retinal cell inducing medium. An exemplary non-limiting medium isDulbecco's Modified Eagle's Medium (DMEM) and F12 at a ratio of about1:1 in the absence of serum. Other tissue culture media can also beused, such as Knock out DMEM. In some embodiments, the cells arecultured for about 36 to about 50 hours, such as for about 48 hours.Additional exemplary media are disclosed in the examples section.

The Wnt pathway inhibitors can be, for example,N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide (CK1-7),3,5,7,8-Tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidin-4-one(XAV939), Secreted frizzled-related protein (SFRP) 1, sFRP1 (GENBANK®Accession No. AF001900.1), sFRP2 (GENBANK® Accession No. NM_003013.2),sFRP3 (GENBANK® Accession No. U91903.1), sFRP4 (GENBANK® Accession No.NM_003014.3), and sFRP5 (GENBANK® Accession No. NM_003015.3,), SFRP-3,SFRP-4 or SFRP-5. These GENBANK® sequences are incorporated herein byreference as available on Jan. 1, 2013. The SFRP can be included at aconcentration of about 5 ng/ml to 100 ng/ml, such as a out 10 ng/ml toabout 90 mg/ml, such as about 20 ng/ml to about 80 ng/ml. In someembodiments, the first medium includes about 3 to about 10 mM of CK1-7dichloride (N-(2-Aminoethyl)-5-chloroisoquinoline-8-sulphonamidedihydrochloride), for example about 3.5 to about 9 mM of CK1-7, or about4 to about 8 mM of CK1-7.

The Nodal pathway inhibitor can be, for example,4-(5-Benzol[1,3]dioxol-5-yl-4-pyridine-2-yl-1H-imidazol-2-yl)-benzamidehydrate,4-[4-(1,3-Benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamidehydrate,4-[4-(3,4-Methylenedioxyphenyl)-5-(2-pyridyl)-1H-imidazol-2-yl]-benzamidehydrate (SB-431542), left-right determination factor (Lefty) or2-(5-Benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridinehydrochloride hydrate (SB-505124). In some embodiments, the first mediumincludes about 3 to about 10 mM of SB43152, for example about 3.5 toabout 9 mM of SB43152, or about 4 to about 8 mM of SB43152.

The cells are cultured in the first medium for about 36 to 72 hours,such as for about 48 hours. Following culture in the first media, suchas for about 36 to about 72 hours, such as for about 48 hours, the EBsare plated on a tissue culture substrate in a second medium. In someembodiments, the tissue culture substrate is coated with MATRIGEL®. Thesecond medium does not include exogenous beta fibroblast growth fact(bFGF). The second media also includes a basic fibroblast growth factor(bFGF) inhibitor, two Wnt pathway inhibitors, and a Nodal pathwayinhibitor. Exemplary media are disclosed in the examples section.

Suitable Wnt pathway inhibitors and Nodal inhibitors are disclosedabove. Suitable bFGF inhibitors include, but are not limited to,N-[(2R)-2,3-Dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]-benzamide(PD0325901),N-[2-[[4-(Diethylamino)butyl]amino-6-(3,5-dimethoxyphenyl)pyrido[2,3-d]pyrimidin-7-yl]-N′-(1,1-dimethylethyl)urea(PD173074), 2-(2-Amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059),1-tert-Butyl-3-[6-(2,6-dichlorophenyl)-2-[[4-(diethylamino)butyl]amino]pyrido[2,3-d]pyrimidin-7-yl]urea(PD161570), or6-(2,6-Dichlorophenyl)-2-[[4-[2-(diethylamino)ethoxy]phenyl]amino]-8-methyl-pyrido[2,3-d]pyrimidin-7(8H)-onedihydrochloride hydrate (PD166285). In some embodiments, the secondmedium comprises about 0.2 to about 2.5 mM of a PD0325901, such as about0.5 to about 2 mM of PD0325901, such as about 1 to about 2 mM ofPD0325901. In one specific non-limiting example, the second mediumincludes about 3.5 to about 9 mM of CK1-7 and about 3.5 to about 9 mM ofSB431542, such as about 5 mM of SB431542.

The second medium can include about 20 to about 90 ng of Noggin, such asabout 30 to about 90 ng of Noggin, such as about 40 to about 80 ng ofNoggin, such as about 50 ng/ml of Noggin.

The second medium can also include about 0.5% to about 3.5% such asabout 1 to about 3%, such as about 2 to about 3%, KNOCKOUT™ serumreplacement to form. KNOCKOUT™ serum replacement is disclosed, forexample, in published U.S. Patent Application No. 2002/076747 and PCTPublication No. 98/830679, which are both incorporated herein byreference, and is available commercially from LIFE TECHNOLOGIES™ toproduced differentiating retinal pigment epithelial cells.

Inhibitors of basic fibroblast growth factor (bFGF, also known as FGF-2)include, but are not limited to,N-[(2R)-2,3-Dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]-benzamide(PD0325901),N-[2-[[4-(Diethylamino)butyl]amino-6-(3,5-dimethoxyphenyl)pyrido[2,3-d]pyrimidin-7-yl]-N′-(1,1-dimethylethyl)urea(PD173074), 2-(2-Amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059),1-tert-Butyl-3-[6-(2,6-dichlorophenyl)-2-[[4-(diethylamino)butyl]amino]pyrido[2,3-d]pyrimidin-7-yl]urea(PD161570), or6-(2,6-Dichlorophenyl)-2-[[4-[2-(diethylamino)ethoxy]phenyl]amino]-8-methyl-pyrido[2,3-d]pyrimidin-7(8H)-onedihydrochloride hydrate (PD166285). In some embodiments, the secondmedium includes about 0.5 to about 2 mM of PD0325901, such as about 1 toabout 2 mM of PD0325901, such as about 1.5 to about 2 mM of PD0325901,such as about 1 mM of PD0325901.

In some embodiments, the cells are cultured in the second medium forabout 18 to about 24 days, such as for about 20 to about 22 days, suchas for about 18, 19, 20, 21, 22, 23 or 24 days. In other embodiments,the cells are cultured in the second medium for a period of about 14days to about four weeks, such about 14 days to about three weeks, suchfor about two weeks, for about 14, about 15, about 16, about 17, about18, about 19, about 20, or about 21 days. Specific non-limiting examplesare for 14 days to three weeks, one week to two weeks, one week to 10days, one week to three weeks, or one week, two weeks or three weeks. Inthis context, “about” indicates within one day of the listed time.

Specific non-limiting examples of exemplary concentrations of PD325901,Noggin and/or DKK1 are shown in FIGS. 8-10. In some embodiments, themethods provide an increase in Pax6 expression in cells that are culturein the second medium for about one to about two weeks, as compared tothe initial population of cells. In certain embodiments, the cells areculture in the absence of, and in the presence of a bFGF inhibitor, andin the presence of Noggin. Non-limiting examples include about 10 mMPD0325901 and about 50 to about 100 ng/ml of Noggin. Additional examplesare presented in FIG. 8. Optionally, the medium can include DKK1 atabout 50 to 100 ng/ml, such as about 75 m 80, 85, 90, 95 or 100 ng/ml ofDKK1. In some embodiments, Pax6 expression is increased at least 150,160, 170, 180, 190, 200, 210, 220, 230, 240 times following culture forone week, as compared to the starting cells. In other embodiments, Pax6expression is increased at least 400, 450, 500, or 600 times followingculture for one to two weeks, as compared to expression in the startingcells. In yet other embodiments, Pax6 expression is increased at least500, 600, 700, 800, or 900 times following culture for one to threeweeks, as compared to expression in the cells prior to culture in thestarting cells, such as embryoid bodies at day zero of culture.

In yet other embodiments, Microphthalmia associated transcription factor(MITF, GENBANK Accession No. NG_011631.1, as available Jan. 1, 2013,incorporated herein by reference) expression is increased at least 4, 5,6, 7, 8, 9 or 10 times following culture for one week, as compared toexpression in the cells prior to culture in the second medium. In otherembodiments, MITF expression is increased at least 3, 4, 5, 6, 7, 8, 9,or 10 times following culture for one to two weeks, as compared toexpression in the cells prior to culture in the second medium. In yetother embodiments, MITF expression is increased at least 6, 7, 8, 9, 10,20, 30, 40, 45, or 50 times following culture for one to three weeks, ascompared to expression in the starting cells, such as embryoid bodies atday 0 of culture.

In some embodiments, expression from a tyrosinase enhancer promoter isincreased. Thus, in some embodiments, expression of a nucleic acidencoding a marker operably linked to the tyrosinase enhancer isincreased. For example, the expression of a marker can be increased byat least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.

The resultant differentiating RPE cells are then transferred to a thirdmedium. In some embodiments, the third medium includes about 50 to about300 ng/ml of ACTIVIN A, such as about 100 to about 200 ng/ml of ACTIVINA, such as about 150 to about 200 ng/ml of ACTIVIN A, such as about 150ng/ml of Activin A. In additional embodiments, the third mediumcomprises about 75 to 150 ng/ml of Wnt3a, such as about 100 to 150 ng/mlof Wnt 3a, such as about 110 to 140 ng/ml of Wnt 3a, such as about 100ng/ml. In this context, about means within 2 ng/ml.

In some embodiments, the cells are cultured in the third medium forabout 18 to about 24 days, such as for about 20 to about 22 days, suchas for about 18, 19, 20, 21, 22, 23 or 24 days. In some embodiments, thecells are cultured in the third medium for a period of about 14 days toabout four weeks, such about 14 days to about three weeks, such forabout two weeks, for about 14, about 15, about 16, about 17, about 18,about 19, about 20, or about 21 days. Specific non-limiting examples arefor 14 days to three weeks, one week to two weeks, one week to 10 days,one week to three weeks, or one week, two weeks or three weeks. In thiscontext, “about” indicates within one day of the listed time.

After culture in the third medium the cells are cultured in a fourthmedium that includes about 3 to about 6 percent serum, such as about 5percent serum. In some embodiments, the serum is fetal calf serum. Inother embodiments, the serum is human serum. The RPE medium includes acanonical WNT inhibitor, a non-canonical WNT inducer, and inhibitors ofthe Sonic and FGF pathway to produce differentiated RPE cells.

This medium also includes a canonical WNT inhibitor, a non-canonical WNTinducer, and inhibitors of the Sonic and FGF pathways. In additionalembodiments, the canonical WNT inhibitor is Dickkopf-related protein.This, in some examples, the medium includes about 50 to about 200 ng/mlof Dickkopf-related protein 1 (DKK1), such as about 50 to about 100ng/ml of DKK1, such as about 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90or 100 ng/ml of DKK1. In some embodiments, the non-canonical WNT induceis Want 5a. In one example, the fourth medium comprises about 50 toabout 200 ng/ml of the non-cannonical WNT inducer, such as WNT5a, suchas about 50 to about 100 ng/ml, such as about 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90 or 100 ng/ml. In another example, the fourth mediumcomprises about 5 to about 20 μM Cycolopamine, such as about 5-10 μMCyclopamine, such as about 5, 6, 7, 8, 9, or 10 μM Cyclopamine. In yetanother embodiments, the fourth medium comprises about 5 to about 20 μMSU5402, such as about 5-10 μM SU5402, such as about 5, 6, 7, 8, 9, or 10μM SU5402.

The cells can be culture in the fourth medium for about 10 to about 20days, such as for about 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 days,such as for about two weeks, or for about 12 to about 16 days.

In some embodiments, the cells are also cultured in the fourth mediumcomprising 3 to about 6% serum, such as about 3% to about 6% human serumor fetal calf serum, such as about 3%, 4%, 5% or 6% fetal calf serum.

Retinal pigment epithelial cells produced by the methods disclosedherein are cultured in the fourth medium containing aphidicolin for 6-8weeks. In some embodiments, the retinal pigment epithelia cells arecultured, for example, in a tissue culture medium including aphidicolinat a concentration of about 3 μM to about 10 μM. In additionalembodiments, the retinal pigment epithelial cells are cultured in atissue culture medium including about 3 μM, about 4 μM, about 5 μM,about 6 μM, about 7 μM, about 8 μM, about 9 μM or about 10 μMaphidicolin. The retinal pigment epithelial cells can be cultured forabout 4 to about 6 weeks in the tissue culture medium includingaphidicolin, such as for about 4 weeks, about 5 weeks, or about 6 weeks.In some embodiments, the use of aphidicolin is sufficient to increasepolarization of the retinal pigment epithelial cells.

The methods disclosed herein efficiently produce RPE cells. Thus, usingthe methods disclosed herein, at least about 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% of the resultant cells are RPE cells. Insome embodiments, a construct including the tyrosinase enhancer operablylinked to a marker, such as a fluorescent protein, is included in theiPSCs. Using the methods disclosed herein, at least about 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the cells express thismarker.

Following culture in the fourth medium the RPE cells can be maintainedin culture. In some embodiments, the RPE cells are maintained in RPEmedium (see above) including comprising about 3 to about 6% serum, suchas about 3%, about 4%, about 5% or about 6% serum. The serum can befetal serum. In some non-limiting examples, the serum is fetal calfserum. In other embodiment, the serum is human serum. The RPE cells canbe maintained in RPE medium, comprising about 3% to about 6% serum, suchas about 5% serum, for example about 5% fetal serum, for about six toabout eight weeks. In some embodiments, the RPE cells are grown intranswells, such as in a 6-well, 12-well, 24-well, or 10 cm plate. Theretinal pigment epithelial cells can be maintained in retinal pigmentepithelial cell (RPE) medium comprising about 5% fetal serum for aboutfour to about ten weeks, such as for about six to about eight weeks,such as for six, seven or eight weeks.

The method can also include confirming that the resultant cells are RPEcells. Method for this confirmation are disclosed below.

Pharmaceutical Compositions and Use of RPE Cells

Disclosed herein are compositions, such as pharmaceutical compositions,including human RPE cells. In certain embodiments, the preparation is apreparation of iPSC-derived RPE cells, such as, but not limited to RPEcells derived from an iPSC produced from a fetal RPE cell, such as ahuman fetal RPE cell. In certain embodiments, these compositions aresubstantially purified (with respect to non-RPE cells) preparationscomprising differentiated RPE cells produced by the methods disclosedherein. These substantially purified populations are compositions thatinclude at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, oreven greater than 99% RPE cells. Thus, the compositions contains lessthan 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than 1% of cells thatare not RPE cells. In certain embodiments, the compositions include atleast about 1×10³ RPE cells, about 1×10⁴ RPE cells, about 1×10⁵ RPEcells, about 1×10⁶ RPE cells, about 1×10⁷ RPE cells, about 1×10⁸ RPEcells, or about 1×10⁹ RPE cells.

In certain embodiments, the cells express one or more of the geneslisted in Table 1 below.

TABLE 1 Gene Symbo Refseq # SLC9A3R1 NM_004252 SLC6A6 NM_003043 SLC12A2NM_001046 SNAI3 NM_178310 SNAI2 NM_003068 SNAI1 NM_005985 CDKN1BNM_004064 PTK2 NM_005607 UTP3 NM_020368 USO1 NM_003715 BHLHE41 NM_030762HSPA13 NM_006948 AKIRIN1 NM_024595 NCRNA00153 NM_018474 FAM13A NM_014883FRMD7 NM_194277 HLA-DRB4 NM_021983 LYZ NM_000239 RGS1 NM_002922 IMPG1NM_001563 PDE6H NM_006205 UNC119 NM_005148 LEPR NM_002303 LGI1 NM_005097HMGCS2 NM_005518 RBP4 NM_006744 OGN NM_014057 RARRES1 NM_002888 RHONM_000539 CHN1 NM_001822 CFH NM_000186 CHI3L1 NM_001276 MAK NM_005906DDC NM_000790 S100A8 NM_002964 BMP5 NM_021073 ITGBL1 NM_004791 PLA2G2ANM_000300 RNASE6 NM_005615 HLA-DRA NM_019111 MS4A4A NM_024021 KLF9NM_001206 PPEF2 NM_006239 LMOD1 NM_012134 HLA-DQB1 NM_002123 IL1R2NM_004633 ASPH NM_004318 MYO6 NM_004999 GPX3 NM_002084 IL33 NM_033439GAP43 NM_002045 ACSL3 NM_004457 FHIT NM_002012 FBLN1 NM_001996 IL6STNM_002184 CPEB3 NM_014912 SPP1 NM_000582 MNDA NM_002432 PSD3 NM_015310SLC16A3 NM_004207 LRAT NM_004744 TMEM176B NM_014020 IGHA1 XM_370781 HEG1NM_020733 KCNV2 NM_133497 SERPINA3 NM_001085 PDE6G NM_002602 PDE6DNM_002601 CD163 NM_004244 RBP3 NM_002900 CFD NM_001928 CXCR4 NM_003467EHHADH NM_001966 NPL NM_030769 NR1D1 NM_021724 RORB NM_006914 MREGNM_018000 VNN2 NM_004665 IGJ NM_144646 CPE NM_001873 RAPGEF4 NM_007023MAFB NM_005461 RCVRN NM_002903 EDNRB NM_000115 S100A9 NM_002965 TOB1NM_005749 SUSD5 NM_015551 SLC25A24 NM_013386 SAG NM_000541 CDKN1CNM_000076 ACSL1 NM_001995 OPN1LW NM_020061 TOX3 NM_00108043 MEGF9NM_00108049 LYVE1 NM_006691 TMEM127 NM_017849 FXYD3 NM_005971 CFINM_000204 TNPO1 NM_002270 C4orf31 NM_024574 HPGD NM_000860 CLUL1NM_199167 EZH1 NM_001991 TOB2 NM_016272 TRPC1 NM_003304 KCND2 NM_012281TRPC4 NM_016179 SOX11 NM_003108 IGF2BP3 NM_006547 RELN NM_005045 HSD17B2NM_002153 FGFR3 NM_000142 ASPM NM_018136 CYTL1 NM_018659 ELN NM_000501BARD1 NM_000465 TRO NM_016157 PTH NM_000315 SERPINH1 NM_001235 TMEFF1NM_003692 COL9A3 NM_001853 C5orf13 NM_004772 PSPH NM_004577 NID2NM_007361 COL11A1 NM_080629 LIPG NM_006033 APC NM_000038 ADAMTSL3NM_207517 TSPAN12 NM_012338 PTPRD NM_002839 HOMER1 NM_004272 MAB21L2NM_006439 TYR NM_000372 TNNC1 NM_003280 CLGN NM_004362 PRMT7 NM_019023PTPLA NM_014241 MFAP2 NM_002403 GLRB NM_000824 TRPM3 NM_020952 PXDNNM_012293 CYP27A1 NM_000784 KDELC1 NM_024089 PDGFC NM_016205 RASGRP3NM_170672 NUP93 NM_014669 SMC6 NM_024624 C11orf9 NM_013279 WFDC1NM_021197 CXorf57 NM_018015 HBG1 NM_000559 PIK3C3 NM_002647 CTSL2NM_001333 NOTCH2NL NM_203458 KCNAB1 NM_003471 SLC5A3 NM_006933 ABHD2NM_007011 SGMS1 NM_147156 GOLGA1 NM_002077 SFRP1 NM_003012 TFECNM_012252 LRRC1 NM_018214 CAPN3 NM_173090 FLRT2 NM_013231 PNPLA3NM_025225 TRIM36 NM_018700 NBEA NM_015678 DAAM1 NM_014992 PLCE1NM_016341 PPFIBP2 NM_003621 MITF NM_000248 NELL2 NM_006159 SC4MOLNM_006745 PLAG1 NM_002655 IGF2BP2 NM_006548 SIX3 NM_005413 CDH3NM_001793 DZIP1 NM_198968 FOXD1 NM_004472 WWTR1 NM_015472 GJA1 NM_000165PLCB4 NM_000933 SEMA3C NM_006379 PKNOX2 NM_022062 COL8A2 NM_005202 WWC2NM_024949 DMXL1 NM_005509 GAS1 NM_002048 GPR143 NM_000273 DCT NM_001922NAV3 NM_014903 SMAD6 NM_005585 CDH1 NM_004360 ASAH1 NM_004315 RAB38NM_022337 PAK1IP1 NM_017906 NOL8 NM_017948 CDO1 NM_001801 PHACTR2NM_014721 SILV NM_006928 TTLL4 NM_014640 MANEA NM_024641 PDPN NM_006474FADS1 NM_013402 HSP90B1 NM_003299 PTPRG NM_002841 VEGFA NM_003376 EFHC1NM_018100 SULF1 NM_015170 GPNMB NM_002510 SDC2 NM_002998 CSPG5 NM_006574MED8 NM_201542 GULP1 NM_016315 MAB21L1 NM_005584 SCAMP1 NM_004866 SLC4A2NM_003040 USP34 NM_014709 FGFR2 NM_000141 SLC6A15 NM_182767 LOXL1NM_005576 SORBS2 NM_003603 LIN7C NM_018362 GEM NM_005261 GPM6B NM_005278APLP1 NM_005166 PITPNA NM_006224 ITGAV NM_002210 RBP1 NM_002899 STAM2NM_005843 TRPM1 NM_002420 NRIP1 NM_003489 ENPP2 NM_006209 RRAGDNM_021244 CHRNA3 NM_000743 SLC6A20 NM_020208 SERPINF1 NM_002615 IFT74NM_025103 LHX2 NM_004789 ALDH1A3 NM_000693 MAP9 NM_00103958 SFRP5NM_003015 SGK3 NM_013257 CLCN4 NM_001830 MFAP3L NM_00100955 BEST1NM_004183 SOSTDC1 NM_015464 BMP4 NM_130851 MET NM_000245 SLC16A4NM_004696 DUSP4 NM_057158 FRZB NM_001463 MYRIP NM_015460 TFPI2 NM_006528TTR NM_000371 TYRP1 NM_000550 RPE65 NM_000329 LIMCH1 NM_014988 SPASTNM_199436 OSTM1 NM_014028 CYP20A1 NM_177538 ATF1 NM_005171 SIL1NM_022464 MPDZ NM_003829 SEPT8 NM_00109881 DCUN1D4 NM_015115 PDZD8NM_173791 LAMP2 NM_002294 DEGS1 NM_003676 DHPS NM_001930 MBNL2 NM_144778DIXDC1 NM_033425 NUDT4 NM_199040 PTGDS NM_000954 CALU NM_001219 RBM34NM_015014 NEDD4L NM_015277 RHOBTB3 NM_014899 NDC80 NM_006101 ARMC9NM_025139 PRNP NM_183079 AHR NM_001621 UBL3 NM_007106 ZNF19 NM_006961RNF13 NM_183384 DAP3 NM_004632 CTBP2 NM_022802 KLHL24 NM_017644 TAX1BP1NM_006024 BDH2 NM_020139 PSME4 NM_014614 GOLPH3L NM_018178 SLC16A1NM_003051 PLOD2 NM_182943 SLC39A6 NM_012319 DNAJB14 NM_024920 LAPTM4BNM_018407 COX15 NM_004376 SMC3 NM_005445 ADAM9 NM_003816 ARL6IP1NM_015161 FAM18B NM_016078 MPHOSPH9 NM_022782 BAT2D1 NM_015172 WASLNM_003941 KLHL21 NM_014851 TIMP3 NM_000362 GRAMD3 NM_023927 LGALS8NM_006499 BCLAF1 NM_014739 PCYOX1 NM_016297 EID1 NM_014335 LSR NM_015925ITM2B NM_021999 ADCY9 NM_001116 CRIM1 NM_016441 EFEMP1 NM_004105 ANKRD12NM_015208 RDH11 NM_016026 CRX NM_000554 SLC24A1 NM_004727 PAX6 NM_000280OTX2 NM_021728 KRT8 NM_002273 RLBP1 NM_000326 MERTK NM_006343 MLANANM_005511 RAB27A NM_183236 OCA2 NM_000275 KCNJ13 NM_002242 CFTRNM_000492 CLDN19 NM_148960 CLDN10 NM_182848 CLDN16 NM_006580 BSGNM_001728 COL4A3 NM_000091 FNDC5 NM_153756 ABCC8 NM_000352 CLCN2NM_004366 SOX2 NM_003106 KLF4 NM_004235 CCND1 NM_053056 RDH5 NM_002905COL8A1 NM_001850 COL9A1 NM_001851 KRT5 NM_000424 PTCH1 NM_000264 SLC7A5NM_003486 SLC2A1 NM_006516 IGFBP5 NM_000599 KRT6B NM_005555 CTNND2NM_001332 SOX9 NM_000346 KCNJ10 NM_002241 KCNJ11 NM_000525 KCNA2NM_004974 KCNB1 NM_004975 CACNA1B NM_000718 CRYAB NM_001885 B2MNM_004048 HPRT1 NM_000194 RPL13A NM_012423 GAPDH NM_002046 ACTBNM_001101 HGDC SA_00105 SLC12A1 NM_000338 MYC NM_002467

The cells can express at least 50, 100, 150, 200, 250, 300, 350, 360,370 or all of these genes. In some embodiments, the cells express MITF(GENBANK® Accession No. NM_000248), PAX6 (GENBANK® Accession No.NM_000280), LHX2 (GENBANK® Accession No. NM_004789), TFEC (GENBANK®Accession No. NM_012252), CDH1 (GENBANK® Accession No. NM_004360), CDH3(GENBANK® Accession No. NM_001793), CLDN10 (GENBANK® Accession No.NM_182848), CLDN16 (GENBANK® Accession No. NM_006580), CLDN19 (GENBANK®Accession No. NM_148960), BEST1 (GENBANK® Accession No. NM_004183),TIMP3 (GENBANK® Accession No. NM_000362), TRPM1 (GENBANK® Accession No.NM_002420), TRPM3 (GENBANK® Accession No. NM_020952), TTR (GENBANK®Accession No. NM_000371), VEGFA (GENBANK® Accession No. NM_003376),CSPG5 (GENBANK® Accession No. NM_006574), DCT (GENBANK® Accession No.NM_001922), TYRP1 (GENBANK® Accession No. NM_000550), TYR (GENBANK®Accession No. NM_000372), SILV (GENBANK® Accession No. NM_006928), SIL1(GENBANK® Accession No. NM_022464), MLANA (NM_005511), RAB27A (GENBANK®Accession No. NM_183236), OCA2 (GENBANK® Accession No. NM_000275),GPR143 (GENBANK® Accession No. NM_000273), GPNMB (GENBANK® Accession No.NM_002510), MYO6 (GENBANK® Accession No. NM_004999), MYRIP (GENBANK®Accession No. NM_015460), RPE65 (GENBANK® Accession No. NM_000329), RBP1(GENBANK® Accession No. NM_002899), RBP4 (GENBANK® Accession No.NM_006744), RDH5 (GENBANK® Accession No. NM_002905), RDH11 (GENBANK®Accession No. NM_016026), RLBP1 (GENBANK® Accession No. NM_000326),MERTK (GENBANK® Accession No. NM_006343), ALDH1A3 (GENBANK® AccessionNo. NM_000693), FBLN1 (GENBANK® Accession No. NM_001996), SLC16A1(GENBANK® Accession No. NM_003051), KCNV2 (GENBANK® Accession No.NM_133497), KCNJ13 (GENBANK® Accession No. NM_002242), and CFTR(GENBANK® Accession No. NM_000492). In other embodiments, the RPE cellsexpress 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 38, 39, 40 or all of these proteins. The GENBANK® disclosuresare incorporated by reference herein, as available on Jan. 31, 2013.

In some embodiments, the RPE cells express MITF, PAX6, LHX2, TFEC, CDH1,CDH3, CLDN10, CLDN16, CLDN19, BEST1, TIMP3, TRPM1, TRPM3, TTR, VEGFA,CSPG5, DCT, TYRP1, TYR, SILV, SIL1, MLANA, RAB27A, OCA2, GPR143, GPNMB,MYO6, MYRIP, RPE65, RBP1, RBP4, RDH5, RDH11, RLBP1, MERTK, ALDH1A3,FBLN1, SLC16A1, KCNV2, KCNJ13, and CFTR.

In further embodiments, the RPE cells also express one or moremicroRNAs. In specific non-limiting examples, the cells express one ormore of microRNAs listed in Table 2.

TABLE 2 Precursor Precursor Mature Mature miRNA miRNA Precursor miRNAmiRNA Position Query Accession Sanger ID Version Accession Sanger ID 1hsa-miR-9* MI0000466 hsa-mir-9-1 14 MIMAT0000442 hsa-miR-9* 2hsa-miR-99a MI0000101 hsa-mir-99a 14 MIMAT0000097 hsa-miR-99a 3hsa-miR-105 MI0000111 hsa-mir-105-1 14 MIMAT0000102 hsa-miR-105 4hsa-miR-107 MI0000114 hsa-mir-107 14 MIMAT0000104 hsa-miR-107 5hsa-miR-125b MI0000446 hsa-mir-125b-1 14 MIMAT0000423 hsa-miR-125b 6hsa-miR-129* MI0000252 hsa-mir-129-1 14 MIMAT0004548 hsa-miR-129* 7hsa-miR-152 MI0000462 hsa-mir-152 14 MIMAT0000438 hsa-miR-152 8hsa-miR-184 MI0000481 hsa-mir-184 14 MIMAT0000454 hsa-miR-184 9hsa-miR-187 MI0000274 hsa-mir-187 14 MIMAT0000262 hsa-miR-187 10hsa-miR-198 MI0000240 hsa-mir-198 14 MIMAT0000228 hsa-miR-198 11hsa-miR-200a MI0000737 hsa-mir-200a 14 MIMAT0000682 hsa-miR-200a 12hsa-miR-200b MI0000342 hsa-mir-200b 14 MIMAT0000318 hsa-miR-200b 13hsa-miR-203 MI0000283 hsa-mir-203 14 MIMAT0000264 hsa-miR-203 14hsa-miR-204 MI0000284 hsa-mir-204 14 MIMAT0000265 hsa-miR-204 15hsa-miR-205 MI0000285 hsa-mir-205 14 MIMAT0000266 hsa-miR-205 16hsa-miR-211 MI0000287 hsa-mir-211 14 MIMAT0000268 hsa-miR-211 17hsa-miR-221 MI0000298 hsa-mir-221 14 MIMAT0000278 hsa-miR-221 18hsa-miR-222 MI0000299 hsa-mir-222 14 MIMAT0000279 hsa-miR-222 19hsa-miR-302b MI0000772 hsa-mir-302b 14 MIMAT0000715 hsa-miR-302b 20hsa-miR-9 MI0000466 hsa-mir-9-1 14 MIMAT0000441 hsa-miR-9 21 hsa-miR-34bMI0000742 hsa-mir-34b 14 MIMAT0004676 hsa-miR-34b 22 hsa-miR-96MI0000098 hsa-mir-96 14 MIMAT0000095 hsa-miR-96 23 hsa-miR-135bMI0000810 hsa-mir-135b 14 MIMAT0000758 hsa-miR-135b 24 hsa-miR-138MI0000476 hsa-mir-138-1 14 MIMAT0000430 hsa-miR-138 25 hsa-miR-149MI0000478 hsa-mir-149 14 MIMAT0000450 hsa-miR-149 26 hsa-miR-181aMI0000289 hsa-mir-181a-1 14 MIMAT0000256 hsa-miR-181a 27 hsa-miR-181bMI0000270 hsa-mir-181b-1 14 MIMAT0000257 hsa-miR-181b 28 hsa-miR-182MI0000272 hsa-mir-182 14 MIMAT0000259 hsa-miR-182 29 hsa-miR-183MI0000273 hsa-mir-183 14 MIMAT0000261 hsa-miR-183 30 hsa-miR-126MI0000471 hsa-mir-126 14 MIMAT0000445 hsa-miR-126 31 hsa-miR-127-3pMI0000472 hsa-mir-127 14 MIMAT0000446 hsa-miR-127-3p 32 hsa-miR-127-5pMI0000472 hsa-mir-127 14 MIMAT0004604 hsa-miR-127-5p 33 hsa-miR-134MI0000474 hsa-mir-134 14 MIMAT0000447 hsa-miR-134 34 hsa-miR-137MI0000454 hsa-mir-137 14 MIMAT0000429 hsa-miR-137 35 hsa-miR-142-3pMI0000458 hsa-mir-142 14 MIMAT0000434 hsa-miR-142-3p 36 hsa-miR-145MI0000461 hsa-mir-145 14 MIMAT0000437 hsa-miR-145 37 hsa-miR-146aMI0000477 hsa-mir-146a 14 MIMAT0000449 hsa-miR-146a 38 hsa-miR-150MI0000479 hsa-mir-150 14 MIMAT0000451 hsa-miR-150 39 hsa-miR-155MI0000681 hsa-mir-155 14 MIMAT0000646 hsa-miR-155 40 hsa-miR-214MI0000290 hsa-mir-214 14 MIMAT0000271 hsa-miR-214 41 hsa-miR-223MI0000300 hsa-mir-223 14 MIMAT0000280 hsa-miR-223 42 hsa-miR-323-3pMI0000807 hsa-mir-323 14 MIMAT0000755 hsa-miR-323-3p 43 hsa-miR-323-5pMI0000807 hsa-mir-323 14 MIMAT0004696 hsa-miR-323-5p 44 hsa-miR-17MI0000071 hsa-mir-17 14 MIMAT0000070 hsa-miR-17 45 hsa-miR-18a MI0000072hsa-mir-18a 14 MIMAT0000072 hsa-miR-18a 46 hsa-miR-19a MI0000073hsa-mir-19a 14 MIMAT0000073 hsa-miR-19a 47 hsa-miR-20a MI0000076hsa-mir-20a 14 MIMAT0000075 hsa-miR-20a 48 hsa-miR-302a MI0000738hsa-mir-302a 14 MIMAT0000684 hsa-miR-302a 49 hsa-miR-302a* MI0000738hsa-mir-302a 14 MIMAT0000683 hsa-miR-302a* 50 hsa-miR-302b* MI0000772hsa-mir-302b 14 MIMAT0000714 hsa-miR-302b* 51 hsa-miR-302c MI0000773hsa-mir-302c 14 MIMAT0000717 hsa-miR-302c 52 hsa-miR-302c* MI0000773hsa-mir-302c 14 MIMAT0000716 hsa-miR-302c* 53 hsa-miR-367* MI0000775hsa-mir-367 14 MIMAT0004686 hsa-miR-367* 54 hsa-miR-371-3p MI0000779hsa-mir-371 14 MIMAT0000723 hsa-miR-371-3p 55 hsa-miR-371-5p MI0000779hsa-mir-371 14 MIMAT0004687 hsa-miR-371-5p 56 hsa-miR-372 MI0000780hsa-mir-372 14 MIMAT0000724 hsa-miR-372 57 hsa-miR-373 MI0000781hsa-mir-373 14 MIMAT0000726 hsa-miR-373 58 hsa-miR-373* MI0000781hsa-mir-373 14 MIMAT0000725 hsa-miR-373* 59 hsa-miR-199b-3p MI0000282hsa-mir-199b 14 MIMAT0004563 hsa-miR-199b-3p 60 hsa-Let-7b MI0000063hsa-let-7b 14 MIMAT0000063 hsa-let-7b 61 hsa-Let-7c MI0000064 hsa-let-7c14 MIMAT0000064 hsa-let-7c 62 hsa-Let-7d MI0000065 hsa-let-7d 14MIMAT0000065 hsa-let-7d 63 hsa-Let-7g MI0000433 hsa-let-7g 14MIMAT0000414 hsa-let-7g 64 hsa-miR-200c MI0000650 hsa-mir-200c 14MIMAT0000617 hsa-miR-200c 65 hsa-miR-147 MI0000262 hsa-mir-147 14MIMAT0000251 hsa-miR-147 66 hsa-miR-429 MI0001641 hsa-mir-429 14MIMAT0001536 hsa-miR-429 67 hsa-miR-124 MI0000443 hsa-mir-124-1 14MIMAT0000422 hsa-miR-124 68 hsa-miR-124* MI0000443 hsa-mir-124-1 14MIMAT0004591 hsa-miR-124* 69 hsa-miR-216a MI0000292 hsa-mir-216a 14MIMAT0000273 hsa-miR-216a 70 hsa-miR-216b MI0005569 hsa-mir-216b 14MIMAT0004959 hsa-miR-216b 71 hsa-miR-139-3p MI0000261 hsa-mir-139 14MIMAT0004552 hsa-miR-139-3p 72 hsa-miR-139-5p MI0000261 hsa-mir-139 14MIMAT0000250 hsa-miR-139-5p 73 hsa-miR-199a-3p MI0000242 hsa-mir-199a-114 MIMAT0000232 hsa-miR-199a-3p 74 hsa-miR-199a-5p MI0000242hsa-mir-199a-1 14 MIMAT0000231 hsa-miR-199a-5p 75 hsa-miR-92a MI0000093hsa-mir-92a-1 14 MIMAT0000092 hsa-miR-92a 76 hsa-miR-92a-1* MI0000093hsa-mir-92a-1 14 MIMAT0004507 hsa-miR-92a-1* 77 hsa-Let-7a MI0000060hsa-let-7a-1 14 MIMAT0000062 hsa-let-7a 78 hsa-Let-7a* MI0000060hsa-let-7a-1 14 MIMAT0004481 hsa-let-7a* 79 hsa-Let-7a-2* MI0000061hsa-let-7a-2 14 MIMAT0010195 hsa-let-7a-2* 80 hsa-miR-455-3p MI0003513hsa-mir-455 14 MIMAT0004784 hsa-miR-455-3p 81 hsa-miR-455-5p MI0003513hsa-mir-455 14 MIMAT0003150 hsa-miR-455-5p 82 hsa-miR-584 MI0003591hsa-mir-584 14 MIMAT0003249 hsa-miR-584 83 hsa-miR-886-5p MI0005527hsa-mir-886 14 MIMAT0004905 hsa-miR-886-5p 84 hsa-miR-34a MI0000268hsa-mir-34a 14 MIMAT0000255 hsa-miR-34a 85 hsa-miR-217 MI0000293hsa-mir-217 14 MIMAT0000274 hsa-miR-217 86 hsa-miR-186 MI0000483hsa-mir-186 14 MIMAT0000456 hsa-miR-186 87 hsa-miR-148a MI0000253hsa-mir-148a 14 MIMAT0000243 hsa-miR-148a 88 hsa-miR-340 MI0000802hsa-mir-340 14 MIMAT0004692 hsa-miR-340 89 SNORD48 NR_002745 SNORD48 90SNORD47 NR_002746 SNORD47 91 SNORD44 NR_002750 SNORD44 92 RNU6-2NR_002752 RNU6-2 93 miRTC N/A miRTC 94 miRTC N/A miRTC 95 PPC N/A PPC 96PPC N/A PPC

Thus, in some embodiments, the cells express at least 2, 5, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95 or all 96 of themiRNAs listed in Table 2. In one specific, non-limiting example, thecells express miR204 and miR211.

Control Genes include, but are not limited to, the genes listed in Table3 below.

TABLE 3 POU5F1 NM_002701 T NM_003181 TF NM_001063 HOXB5 NM_002147 KRT23NM_015515 HOXA4 NM_002141 VSX2 NM_182894 AFP NM_001134 FOXA2 NM_021784SMAD3 NM_005902 NANOG NM_024865

These genes are expressed in RPE cells and in control cells. Thus, insome embodiments, the RPE cells express one or more of these markers.The RPE cells can express 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or all of thesemarkers.

In yet other embodiments the RPE cells have a resting potential of about−50 to about −60 mV, and a fluid transport rate of about 5 to about 10μl cm⁻²h⁻¹. In additional embodiments, the RPE cells express MITF, PAX6,LHX2, TFEC, CDH1, CDH3, CLDN10, CLDN16, CLDN19, BEST1, TIMP3, TRPM1,TRPM3, TTR, VEGFA, CSPG5, DCT, TYRP1, TYR, SILV, SILL MLANA, RAB27A,OCA2, GPR143, GPNMB, MYO6, MYRIP, RPE65, RBP1, RBP4, RDH5, RDH11, RLBP1,MERTK, ALDH1A3, FBLN1, SLC16A1, KCNV2, KCNJ13, and CFTR, express miR204and miR211, have a resting potential of about −50 to about −60 mV andhave a fluid transport rate of about 5 to about 10 μl cm⁻²h⁻¹.

Compositions are also provided that include a scaffold, such as apolymeric carrier and/or an extracellular matrix, and an effectiveamount of the RPE cells disclosed herein. The extracellular matrix canbe a human extracellular matrix. The polymeric particle can be amicroparticle. In some embodiments, the cells are provided as amonolayer.

A variety of biological or synthetic solid matrix materials (i.e., solidsupport matrices, biological adhesives or dressings, andbiological/medical scaffolds) are suitable for use. The matrix materialis generally physiologically acceptable and suitable for use in vivoapplications. Non-limiting examples of such physiologically acceptablematerials include, but are not limited to, solid matrix materials thatare absorbable and/or non-absorbable, such as small intestine submucosa(SIS), e.g., porcine-derived (and other SIS sources); crosslinked ornon-crosslinked alginate, hydrocolloid, foams, collagen gel, collagensponge, polyglycolic acid (PGA) mesh, polyglactin (PGL) mesh, fleeces,and bioadhesives (e.g., fibrin glue and fibrin gel). The polymer can bepoly(DL)-lactic-co-glycolic) acid (PLGA) (see Lu et al., J. Biomater SciPolym Ed. 9(11): 1187-205, 1998). In other embodiments, the matricincludes poly(L-lactic acid) (PLLA) and poly(D,L-lactic-co-glycolicacid) (PLGA) such as with a co-polymer ratio of about 90:10, 75:25,50:50, 25:75, 10:90 (PLLA:PLGA) (see Thomson et al., J. Biomed. MaterRes. A 95: 1233-42, 2010).

Suitable polymeric carriers also include porous meshes or sponges formedof synthetic or natural polymers, as well as polymer solutions. Onenon-limiting form of a matrix is a polymeric mesh or sponge. Anothernon-limiting example is a polymeric hydrogel. Natural polymers that canbe used include proteins such as collagen, albumin, and fibrin; andpolysaccharides such as alginate and polymers of hyaluronic acid.Synthetic polymers include both biodegradable and non-biodegradablepolymers. Examples of biodegradable polymers include polymers of hydroxyacids such as polylactic acid (PLA), polyglycolic acid (PGA), andpolylactic acid-glycolic acid (PLGA), polyorthoesters, polyanhydrides,polyphosphazenes, and combinations thereof. Non-biodegradable polymersinclude polyacrylates, polymethacrylates, ethylene vinyl acetate, andpolyvinyl alcohols.

Polymers that can form ionic or covalently crosslinked hydrogels whichare malleable can be used. A hydrogel is a substance formed when anorganic polymer (natural or synthetic) is crosslinked via covalent,ionic, or hydrogen bonds to create a three-dimensional open-latticestructure which entraps water molecules to form a gel. Examples ofmaterials which can be used to form a hydrogel include polysaccharidessuch as alginate, polyphosphazines, and polyacrylates, which arecrosslinked ionically, or block copolymers such as PLURONICS™ orTETRONICS™, polyethylene oxide-polypropylene glycol block copolymerswhich are crosslinked by temperature or pH, respectively. Othermaterials include proteins such as fibrin, polymers such aspolyvinylpyrrolidone, hyaluronic acid and collagen.

In general, these polymers are at least partially soluble in aqueoussolutions, such as water, buffered salt solutions, or aqueous alcoholsolutions that have charged side groups, or a monovalent ionic saltthereof. Examples of polymers with acidic side groups that can bereacted with cations are poly(phosphazenes), poly(acrylic acids),poly(methacrylic acids), copolymers of acrylic acid and methacrylicacid, poly(vinyl acetate), and sulfonated polymers, such as sulfonatedpolystyrene. Copolymers having acidic side groups formed by reaction ofacrylic or methacrylic acid and vinyl ether monomers or polymers canalso be used. Examples of acidic groups are carboxylic acid groups,sulfonic acid groups, halogenated (preferably fluorinated) alcoholgroups, phenolic OH groups, and acidic OH groups. Examples of polymerswith basic side groups that can be reacted with anions are poly(vinylamines), poly(vinyl pyridine), poly(vinyl imidazole), and some iminosubstituted polyphosphazenes. The ammonium or quaternary salt of thepolymers can also be formed from the backbone nitrogens or pendant iminogroups. Examples of basic side groups are amino and imino groups.

Non-limiting examples of suitable materials for the substrate includeparylene polypropylene, polyimide, glass, nitinol, polyvinyl alcohol,polyvinyl pyrolidone, collagen, chemically-treated collagen,polyethersulfone (PES), poly(glycerol-sebacate) PGS,poly(styrene-isobutyl-styrene), polyurethane, ethyl vinyl acetate (EVA),polyetherether ketone (PEEK), Kynar (Polyvinylidene Fluoride; PVDF),Polytetrafluoroethylene (PTFE), Polymethylmethacrylate (PMMA), Pebax,acrylic, polyolefin, polydimethylsiloxane (PDMS) and other siliconeelastomers, polypropylene, hydroxyapetite, titanium, gold, silver,platinum, other metals and alloys, ceramics, plastics and mixtures orcombinations thereof. Additional suitable materials used to constructcertain embodiments of the substrates include, but are not limited to,poly-para-xylylenes (e.g., parylene, including but not limited toparylene A, parylene AM, parylene C, ammonia treated parylene, paryleneC treated with polydopamine), poly(lactic acid) (PLA),polyethylene-vinyl acetate, poly(lactic-co-glycolic acid) (PLGA),poly(D,L-lactide), poly(D,L-lactide-co-trimethylene carbonate),collagen, heparinized collagen, denatured collagen, modified collagen(e.g., silicone with gelatin), other cell growth matrices (such asSYNTHEMAX™), poly(caprolactone), poly(glycolic acid), and/or otherpolymer, copolymers, or block co-polymers, poly(caprolactone) containingcyclic Arginine-Glycine-Asparagine, cyclic or linearArginine-Glycine-aspartic acid, blends of polycaprolactone andpolyethylene glycol (PCL-PEG), thermoplastic polyurethanes,silicone-modified polyether urethanes, poly(carbonate urethane), orpolyimide. Thermoplastic polyurethanes are polymers or copolymers whichmay comprise aliphatic polyurethanes, aromatic polyurethanes,polyurethane hydrogel-forming materials, hydrophilic polyurethanes, orcombinations thereof. Non-limiting examples include elasthane(poly(ether urethane)) such as ELASTHANE™ 80A, Lubrizol, TECOPHILIC™,PELLETHANE™, CARBOTHANE™, TECOTHANE™, TECOPLAST™, AND ESTANE™.Silicone-modified polyether urethanes may include CARBOSIL™ 20 orPURSIL™ 20 80A, and the like. Poly(carbonate urethane) may includeBIONATE™ 80A or similar polymers. Moreover, in several embodiments thesubstrate (and/or the cells) comprises materials (or chemicals) thatallow for visualization of the substrate in situ, which are unaffectedby the cryopreservation (and thawing) of the substrate and cells

The retinal pigment epithelial cells produced by the methods disclosedherein can be cryopreserved, see for example, PCT Publication No.2012/149484 A2, which is incorporated by reference herein. The cells canbe cryopreserved with or without a substrate. In several embodiments,the storage temperature ranges from about −50° C. to about −60° C.,about −600° C. to about −70° C., about −70° C. to about −80° C., about−80° C. to about −90° C., about −90° C. to about −100° C., andoverlapping ranges thereof. In some embodiments, lower temperatures areused for the storage (e.g., maintenance) of the cryopreserved cells. Inseveral embodiments, liquid nitrogen (or other similar liquid coolant)is used to store the cells. In further embodiments, the cells are storedfor greater than about 6 hours. In additional embodiments, the cells arestored about 72 hours. In several embodiments, the cells are stored 48hours to about one week. In yet other embodiments, the cells are storedfor about 1, 2, 3, 4, 5, 6, 7, or 8 weeks. In further embodiments, thecells are stored for 1, 2, 3, 4, 5, 67, 8, 9, 10, 11 or 12 months. Thecells can also be stored for longer times. The cells can becryopreserved separately or on a substrate, such as any of thesubstrates disclosed herein.

A general method of cryopreserving cells is disclosed herein, that canbe used for cryopreservation of any cell type, such as stem cells,including iPSCs, and differentiated cells, such as retinal pigmentepithelial cells. The method includes the use of alginate. “Alginate”(or “alginic acid” or “algin”) refers to the anionic polysaccharidedistributed widely in the cell walls of brown algae. Alginate formswater-soluble salts with alkali metals, such as sodium, potassium,lithium, magnesium, ammonium, and the substituted ammonium cationsderived from lower amines, such as methyl amine, ethanol amine,diethanol amine, and triethanol amine. Alginate includes calciumalginate, sodium alginate, propylene-glycol alginate, and potassiumalginate.

In some embodiments, cells, such as the disclosed retinal pigmentepithelial cells are contacted with an effective amount of alginate. Thecells are contacted with alginate, and then exposed to divalent cations,such as Calcium, Barium, Copper, Zinc or Strontium) which results incross-linking of the alginate polymers in the cell/liquid alginatesuspension (see for example, U.S. Published Patent Application No.2012/0171295, incorporated herein by reference). In certain embodiments,the divalent cation used to cross-link the alginate in the cell/liquidalginate solution is calcium chloride (CaCl₂), barium chloride (BaCl₂),strontium chloride (SrCl₂), copper chloride (CuCl₂), or zinc chloride(ZnCl₂). In a specific embodiment, the divalent cation used tocross-link the alginate in the cell/liquid alginate solution is calciumchloride (CaCl₂). In certain embodiments, the solution of divalentcation comprises about 0.5%, about 0.75%, about 1.0%, about 1.25%, about1.5%, about 1.75%, or about 2.0% divalent cation. In a specificembodiment, the solution of divalent cation comprises 1.5% divalentcation, e.g., CaCl₂.

In some embodiments, additional cryoprotectants can be used. Forexample, cells can be cryopreserved in a cryopreservation solutioncomprising one or more cryoprotectants, such as DMSO, serum albumin,such as human or bovine serum albumin. In certain embodiments, thesolution comprises about 1%, about 1.5%, about 2%, about 2.5%, about 3%,about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%DMSO. In other embodiments, the solution comprises about 1% to about 3%,about 2% to about 4%, about 3% to about 5%, about 4% to about 6%, about5% to about 7%, about 6% to about 8%, about 7% to about 9%, or about 8%to about 10% DMSO or albumin. In a specific embodiment, the solutioncomprises 2.5% DMSO. In another specific embodiment, the solutioncomprises 10% DMSO.

Cells can be cryopreserved in small containers (e.g., ampoules); in bagssuitable for cryopreservation; or in any other suitable container forcryopreservation. In some embodiments, cells are cryopreserved incommercially available cryopreservation medium. The cells can becryopreserved in a cryopreservation solution comprising one or moresolutions for use in storing cells. Cryopreservation solutions includedCryoStor CS10® and HYPOTHERMOSO1® (BioLife Solutions, Bothell, Wash.)).In certain embodiments, the solution comprises about 25%, about 30%,about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about65%, or about 70% HYPOTHERMOSOL®. In other embodiments, the solutioncomprises about 25% to about 50%, about 40% to about 60%, about 50% toabout 60%, about 50% to about 70%, or about 60% to about 70%HYPOTHERMOSOL®. In a specific embodiment, the solution comprises 55%HYPOTHERMOSOL®. In another specific embodiment, the solution comprises57.5% HYPOTHERMOSOL®. In additional embodiments, the cryopreservationsolution can include one or more excipients, such as dextran, starch,glucose, lactose, sucrose, gelatin, silica gel, glycerol monostearate,sodium chloride, glycerol, propylene, and/or glycol. Thecryopreservation solution can include media, such as the media disclosedherein. In additional embodiments, the medium can be phosphate bufferedsaline or Dulbecco's Modified Eagle's Medium (DMEM).

Cells may be cooled, for example, at about 1° C./minute duringcryopreservation. In some embodiments, the cryopreservation temperatureis about −80° C. to about −180° C., or about −125° C. to about −140° C.In some embodiments, the cells are cooled to 4° C. prior to cooling atabout 1° C./minute. Cryopreserved cells can be transferred to vaporphase of liquid nitrogen prior to thawing for use. In some embodiments,for example, once the cells have reached about −80° C., they aretransferred to a liquid nitrogen storage area. Cryopreservation can alsobe done using a controlled-rate freezer. Cryopreserved cells may bethawed, e.g., at a temperature of about 25° C. to about 40° C., andtypically at a temperature of about 37° C.

The human RPE cells described herein, or a pharmaceutical compositionincluding these cells, can be used for the manufacture of a medicamentto treat a condition in a patient in need thereof. The RPE cells can bepreviously cryopreserved. The disclosed RPE cells are derived fromiPSCs, and thus can be used to provide “personalized medicine” forpatients with eye diseases. In some embodiments, somatic cells obtainedfrom patients can be genetically engineered to correct the diseasecausing mutation, differentiated into RPE, and engineered to form an RPEtissue. This RPE tissue can be used to replace the endogenousdegenerated RPE of the same patient. Alternatively, iPSCs can begenerated from a healthy donor or from HLA homozygous “super-donors” canbe used. RPE cells can be treated in vitro with certain factors, such aspigment epithelium-derived factor (PEDF), transforming growth factor(TGF)-beta, and/or retinoic acid to generate an anti-inflammatory andimmunosuppressive environment in vivo.

Various eye conditions may be treated or prevented by the introductionof the RPE cells obtained using the methods disclosed herein. Theconditions include retinal diseases or disorders generally associatedwith retinal dysfunction or degradation, retinal injury, and/or loss ofretinal pigment epithelium. Conditions that can be treated include,without limitation, degenerative diseases of the retina, such asStargardt's macular dystrophy, retinitis pigmentosa, maculardegeneration (such as age related macular degeneration), glaucoma, anddiabetic retinopathy. Additional conditions include Lebers congenitalamaurosis, hereditary or acquired macular degeneration, Best disease,retinal detachment, gyrate atrophy, choroideremia, pattern dystrophy,other dystrophies of the RPE, and RPE and retinal damage due to damagecaused by any one of photic, laser, inflammatory, infectious, radiation,neovascular or traumatic injury. In certain embodiments, methods areprovided for treating or preventing a condition characterized by retinaldegeneration, comprising administering to a subject in need thereof aneffective amount of a composition comprising RPE cells. These methodscan include selecting a subject with one or more of these conditions,and administering a therapeutically effective amount of the RPE cellssufficient to treat the condition and/or ameliorate symptoms of thecondition. The RPE cells may be transplanted in various formats. Forexample, the RPE cells may be introduced into the target site an theform of cell suspension, or adhered onto a matrix, extracellular matrixor substrate such as a biodegradable polymer, as a monolayer, or acombination. The RPE cells may also be transplanted together(co-transplantation) with other retinal cells, such as withphotoreceptors. In some embodiments, the RPE cells are produced fromiPSCs from the subject to be treated, and thus are autologous. In otherembodiments, the RPE cells are produced from an MHC-matched donor.

The RPE cells can be introduced to various target sites within asubject's eye. In some embodiments, RPE cells are introduced, such as bytransplantation, to the subretinal space of the eye, which is theanatomical location of the RPE (between the photoreceptor outer segmentsand the choroids) in mammals. In addition, dependent upon migratoryability and/or positive paracrine effects of the cells, introductioninto additional ocular compartments can be considered, such as thevitreal space, the inner or outer retina, the retinal periphery andwithin the choroids.

The cells can be introduced by various techniques known in the art.Methods for performing RPE transplants are disclosed in, for example, inU.S. Pat. Nos. 5,962,027, 6,045,791, and 5,941,250; Biochem Biophys ResCommun Feb. 24, 2000; 268(3): 842-6; and Opthalmic Surg February 1991;22(2): 102-8). Methods for performing corneal transplants are describedin, for example, U.S. Pat. No. 5,755,785; Curr Opin Opthalmol August1992; 3 (4): 473-81; Ophthalmic Surg Lasers April 1998; 29 (4): 305-8;and Opthalmology April 2000; 107 (4): 719-24. In some embodiments,transplantation is performed via pars pana vitrectomy surgery followedby delivery of the RPE cells through a small retinal opening into thesub-retinal space or by direct injection. Alternatively, RPE cells canbe delivered into the subretinal space via a trans-scleral,trans-choroidal approach. In addition, direct trans-scleral injectioninto the vitreal space or delivery to the anterior retinal periphery inproximity to the ciliary body can be performed.

The cells can also be incorporated into a delivery device. If mainlyparacrine effects are to be utilized, cells can be delivered andmaintained in the eye encapsulated within a semi-permeable container,which decreases exposure of the cells to the host immune system(Neurotech USA CNTF delivery system; PNAS. 103(10) 3896-3901, 2006).

The RPE cells can be introduced into the target site in the form of cellsuspension, adhered onto a matrix, such as extracellular matrix, orprovided on substrate such as a biodegradable polymer. The RPE cells canalso be transplanted together (co-transplantation) with other cells,such as retinal cells with photoreceptors. Thus, a compositioncomprising RPE cells obtained by the methods disclosed herein isprovided. In some embodiments, these RPE cells include a tyrosinaseenhancer operably linked to a promoter and a nucleic acid encoding amarker. In other embodiments, the RPE cells also include a secondconstitutive promoter operably linked to a nucleic acid encoding asecond marker.

Screening Methods and Identification of RPE Cells

Methods are also provided for the identification of RPE cells, and/orconfirming a cell is an RPE cell. The cells can be produced using themethods disclosed herein.

A method is provided herein for identifying an agent that alters thedifferentiation and/or proliferation of RPE cells. In some embodiments,methods are provided for identifying an agent that alters theproliferation of RPE cells. The methods include contacting an RPE cellwith an effective amount of an agent of interest. In some embodiments,methods are provided for identifying an agent that increasesdifferentiation of RPE.

In additional embodiments, methods are provided for identifying an agentthat affects RPE cell survival, and/or changes the endogenous expressionof genes in RPE cells. Therapeutic agents can be identified for thetreatment of disease using these methods. In some embodiments, methodsare provided for identifying an agent that affects the epithelialphenotype of RPE cells. The method includes contacting iPSCs, RPEs, orembryoid bodies with the agent of interest, and assaying the productionand/or survival and/or phenotype of RPE cells. The agent can beintroduced into any step of the methods disclosed herein.

RPE cells produced using the methods disclosed herein, or other RPEcells, can also be contact with an agent, and assessed using the methodsdisclosed below. These methods can be used to identify agents thataffect expression of genes, or to identify agents that affect survivalof RPE cells. In some embodiments, the RPE cells are treated with astressor, such as thapsigargin, A23187, DL-dithiothreitol, or2-deoxy-D-glucose.

The test compound can be any compound of interest, including chemicalcompounds, small molecules, polypeptides, growth factors, cytokines, orother biological agents (for example antibodies). In several examples, apanel of potential neurotrophic agents are screened. In otherembodiments a panel of polypeptide variants is screened.

In some embodiments, methods are provided for determining if an agent ofinterest increases the differentiation of retinal pigment epithelialcells. The method includes culturing the embryoid bodies produced fromhuman induced pluripotent stem cells comprising a nucleic acid encodinga first marker operably linked to a retinal pigment epithelial cellspecific promoter, and comprising a second marker operably linked to aconstitutive promoter, as disclosed above, in a first medium comprisingtwo Wnt pathway inhibitor and a Nodal pathway inhibitor. The embryoidbodies are plated on a tissue culture substrate in a second medium that(a) does not comprise beta fibroblast growth fact (bFGF) (b) comprises abasic fibroblast growth factor (bFGF) inhibitor, the two Wnt pathwayinhibitors, and the Nodal pathway inhibitor; and (c) comprises about 20to about 90 ng of Noggin to form differentiating retinal pigmentepithelial cells. The differentiating retinal pigment epithelial cellsare cultured in a third medium comprising ACTIVAN A and WNT3a. The cellsare then cultured in a fourth retinal pigment epithelial cell (RPE)medium comprising about 5% fetal serum, a canonical WNT inhibitor, anon-canonical WNT inducer, and inhibitors of the Sonic and FGF pathwayto producing human retinal pigment epithelial cells. Suitable methodsare disclosed herein.

One, several or all of these steps are performed in the presence of theagent of interest. The expression of the first marker in the retinalpigment epithelial cells is compared to the expression of the secondmarker, wherein an increase in expression of the first marker ascompared to the second marker indicates that the agent increases thedifferentiation of retinal pigment epithelial cells

Methods for preparing a combinatorial library of molecules that can betested for a desired activity are well known in the art and include, forexample, methods of making a phage display library of peptides, whichcan be constrained peptides (see, for example, U.S. Pat. Nos. 5,622,699;5,206,347; Scott and Smith, Science 249:386-390, 1992; Markland et al.,Gene 109:13-19, 1991), a peptide library (U.S. Pat. No. 5,264,563); anFDA-approved drug library (see, for example, Huang, E; Southall, N;Wang, Y et al., Science Translational Medicine 3: 1-12); apeptidomimetic library (Blondelle et al., Trends Anal Chem. 14:83-92,1995); a nucleic acid library (O'Connell et al., Proc. Natl Acad. Sci.,USA 93:5883-5887, 1996; Tuerk and Gold, Science 249:505-510, 1990; Goldet al., Ann. Rev. Biochem. 64:763-797, 1995); an oligosaccharide library(York et al., Carb. Res. 285:99-128, 1996; Liang et al., Science274:1520-1522, 1996; Ding et al., Adv. Expt. Med. Biol. 376:261-269,1995); a lipoprotein library (de Kruif et al., FEBS Lett. 3 99:23 2-236, 1996); a glycoprotein or glycolipid library (Karaoglu et al., J CellBiol. 130.567-577, 1995); or a chemical library containing, for example,drugs or other pharmaceutical agents (Gordon et al., J Med. Chem.37.1385-1401, 1994; Ecker and Crooke, BioTechnology 13:351-360, 1995).Polynucleotides can be particularly useful as agents that can alter afunction of cells (such as, but not limited to iPSCs, embryoid bodiesand RPE cells) because nucleic acid molecules having binding specificityfor cellular targets, including cellular polypeptides, exist naturally,and because synthetic molecules having such specificity can be readilyprepared and identified (see, for example, U.S. Pat. No. 5,750,342).

In one embodiment, for a high throughput format, iPSCs, embryoid bodiesor RPE progenitors can be introduced into wells of a multi-well plate orof a glass slide or microchip, and can be contacted with the test agent.Generally, the cells are organized in an array, particularly anaddressable array, such that robotics conveniently can be used formanipulating the cells and solutions and for monitoring the stem orprecursor cells, particularly with respect to the function beingexamined. An advantage of using a high throughput format is that anumber of test agents can be examined in parallel, and, if desired,control reactions also can be run under identical conditions as the testconditions. As such, the methods disclosed herein provide a means toscreen one, a few, or a large number of test agents in order to identifyan agent that can alter a function of cells, for example, an agent thatinduces the cells to differentiate into a desired cell type, or thataffects differentiation, survival and/or cell proliferation. Highthroughput screens can be used to assess phenotype and survival. Thesescreens can be used to identify drugs that can affect RPE phenotype andsurvival. In some embodiments, RPE phenotype is assayed by loss/gain ofa fluorescent signal and survival is assayed (such as by an ATP basedcell titer glow assay).

These methods can include evaluating expression of one or more of thegenes listed in Table 1, Table A or FIG. 27C. In some embodiments, theexpression of 50, 100, 150, 200, 250, 300, 350, 360, 370 or all of thegenes listed in Table 1 can be assessed. For any of these methods, theexpression of one or more of MITF (GENBANK® Accession No. NM_000248),PAX6 (GENBANK® Accession No. NM_000280), LHX2 (GENBANK® Accession No.NM_004789), TFEC (GENBANK® Accession No. NM_012252), CDH1 (GENBANK®Accession No. NM_004360), CDH3 (GENBANK® Accession No. NM_001793),CLDN10 (GENBANK® Accession No. NM_182848), CLDN16 (GENBANK® AccessionNo. NM_006580), CLDN19 (GENBANK® Accession No. NM_148960), BEST1(GENBANK® Accession No. NM_004183), TIMP3 (GENBANK® Accession No.NM_000362), TRPM1 (GENBANK® Accession No. NM_002420), TRPM3 (GENBANK®Accession No. NM_020952), TTR (GENBANK® Accession No. NM_000371), VEGFA(GENBANK® Accession No. NM_003376), CSPG5 (GENBANK® Accession No.NM_006574), DCT (GENBANK® Accession No. NM_001922), TYRP1 (GENBANK®Accession No. NM_000550), TYR (GENBANK® Accession No. NM_000372), SILV(GENBANK® Accession No. NM_006928), SIL1 (GENBANK® Accession No.NM_022464), MLANA (NM_005511), RAB27A (GENBANK® Accession No.NM_183236), OCA2 (GENBANK® Accession No. NM_000275), GPR143 (GENBANK®Accession No. NM_000273), GPNMB (GENBANK® Accession No. NM_002510), MYO6(GENBANK® Accession No. NM_004999), MYRIP (GENBANK® Accession No.NM_015460), RPE65 (GENBANK® Accession No. NM_000329), RBP1 (GENBANK®Accession No. NM_002899), RBP4 (GENBANK® Accession No. NM_006744), RDH5(GENBANK® Accession No. NM_002905), RDH11 (GENBANK® Accession No.NM_016026), RLBP1 (GENBANK® Accession No. NM_000326), MERTK (GENBANK®Accession No. NM_006343), ALDH1A3 (GENBANK® Accession No. NM_000693),FBLN1 (GENBANK® Accession No. NM_001996), SLC16A1 (GENBANK® AccessionNo. NM_003051), KCNV2 (GENBANK® Accession No. NM_133497), KCNJ13(GENBANK® Accession No. NM_002242), and CFTR (GENBANK® Accession No.NM_000492) RNA or protein is assessed. The GENBANK® disclosures areincorporated by reference herein, as available on Jan. 31, 2013.Increased expression of the one or more mRNAs or proteins followingexposure to the agent, as compared to iPSC or embryoid bodies notcontacted with the agent, indicates that the agent affects RPE celldifferentiation, survival and/or proliferation, specifically that itincreases RPE cell differentiation, survival and/or proliferation.Decreased expression of the one or more mRNAs or proteins followingexposure to the agent, as compared to iPSC or embryoid bodies notcontacted with the agent, indicates that the agent affects RPE celldifferentiation, survival and/or proliferation, specifically that itdecreases RPE cell differentiation, survival and/or proliferation.

In other embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 38, 39, 40 or all of these mRNAs or proteins isassessed. Increased expression of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 38, 39, 40 or all of these mRNAs orproteins indicates that the agent increases RPE cell differentiation,survival and/or proliferation. Decreased expression of 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 38, 39, 40 or all ofthese mRNAs or proteins indicates that the agent decreases RPE celldifferentiation, survival and/or proliferation.

In some embodiments, the expression of MITF, PAX6, LHX2, TFEC, CDH1,CDH3, CLDN10, CLDN16, CLDN19, BEST1, TIMP3, TRPM1, TRPM3, TTR, VEGFA,CSPG5, DCT, TYRP1, TYR, SILV, SILL MLANA, RAB27A, OCA2, GPR143, GPNMB,MYO6, MYRIP, RPE65, RBP1, RBP4, RDH5, RDH11, RLBP1, MERTK, ALDH1A3,FBLN1, SLC16A1, KCNV2, KCNJ13, and CFTR is assessed. Increasedexpression indicates that the agent increases RPE cell differentiation,survival or proliferation. Decreased expression indicates that the agentdecreases RPE cell differentiation, survival or proliferation. Theexpression of one or a combination of these markers also can be used toconfirm that a cell is a RPE cell.

In additional embodiments, the expression of at least 2, 5, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95 or all 96 of themiRNAs listed in Table 2 is assessed. In further embodiments, theproduction of miR204 (GENBANK® Accession No. NR_029621.1, Dec. 9, 2012,incorporated herein by reference) and/or miR211 (GENBANK® Accession No.NR_029624.1, Sep. 23, 2012, incorporated herein by reference) isassessed. Increased production of one or both of the microRNAs indicatesthat the agent affects RPE cells differentiation and/or proliferation,or confirms that the cell is an RPE. Increased expression indicates thatthe agent increases RPE cell differentiation, survival or proliferation.Decreased expression indicates that the agent decreases RPE celldifferentiation, survival or proliferation. These miRNAs can be detectedusing the exemplary methods for detecting nucleic acids disclosed above.

In other embodiments, the expression of one or more of the genes listedin Table 3 is also assessed. The expression of these genes can be usedas a control, such as a reference standard. Thus, in some embodiments,the expression of these genes does not change in the presence of theagent of interest. These genes can also be used as reference standards.

In yet other embodiments, expression of one or more of the moleculeslisted in FIG. 27C is evaluated. Thus, in some embodiments, 1, 2, 3, 4,5, 6, 7, 8, 9 or 10 of the molecules listed in FIG. 27C are evaluated.In additional embodiment, expression of one or more of the moleculeslisted in Table A is evaluated. The expression of 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, or 37 of the moleculeslisted in Table A can be evaluated.

In yet other embodiments, the expression of one or more of SOX2 (e.g.,GENBANK® Accession No. NM_003106), PAX6 (e.g., GENBANK® Accession No.NM_001604), RPE65 (e.g., GENBANK® Accession No. NM_000329), RDH5, (e.g.,GENBANK® Accession No. NM_000329), TRPM1 (e.g., GENBANK® Accession No.NM_002420) and BEST1 (e.g., GENBANK® Accession No. NM_004183) isevaluated, all GENBANK® Accession information incorporated by referenceas available on Jan. 31, 2013. In some embodiments, compared toundifferentiated iPSCs, iPSC-derived RPE expresses lower levels ofneural progenitor factor SOX2 and much higher levels of RPE-specificgenes PAX6, RPE65, RDH5, TRPM1, and BEST1.

The methods can include determining if an agent of interest increasesthe differentiation iPSCs into RPE cells. If the agent increases thedifferentiation into RPE cells, the expression of SOX2 is decreased andthe expression of PAX6, RPE65, RDH5, TRPM1, and BEST1 is increased inthe sample as compared to a control. In another embodiment, theexpression of SOX2 is decreased and the expression of two, three, fouror five of PAX6, RPE65, RDH5, TRPM1, and BEST1 are increased in thesample as compared to a control. The control can be a standard value ora sample contacted with an agent known not to increase thedifferentiation into RPE cells. The methods can also include determiningif an agent of interest decreases the differentiation of iPSCs into RPEcells, and thus maintains the iPSCs in an undifferentiated state. If theagent decreases the differentiation into RPE cells, the expression ofSOX2 is increased and the expression of PAX6, RPE65, RDH5, TRPM1, andBEST1 is decreased as compared to a control. In another embodiment, ifthe agent decreased differentiation into RPE cells, the expression ofSOX2 is increased and the expression of two, three, four or five ofPAX6, RPE65, RDH5, TRPM1, and BEST1 are decreased in the sample ascompared to a control. The control can be a standard value or a sampleconstrued with an agent known to increase differentiation into RPEcells. Exemplary non-limiting assays are disclosed in Example 8.

Nucleic acids can be detected by any method known in the art. In someexamples, nucleic acids are isolated, amplified, or both, prior todetection. In an example, cells or a fraction thereof, such as purifiednucleic acids, can be incubated with primers that permit theamplification of one or more of mRNAs, under conditions sufficient topermit amplification of such products. For example, the biologicalsample is incubated with primers or probes that can bind to one or moreof the disclosed nucleic acid sequences (such as cDNA, genomic DNA, orRNA (such as mRNA) under high stringency conditions. The resultinghybridization can then be detected using methods known in the art.

The nucleic acid sample can be amplified. If a quantitative result isdesired, a method is utilized that maintains or controls for therelative frequencies of the amplified nucleic acids. Methods of“quantitative” amplification are well known to those of skill in theart. For example, quantitative PCR involves simultaneously co-amplifyinga known quantity of a control sequence using the same primers. Thisprovides an internal standard that can be used to calibrate the PCRreaction. The array can then include probes specific to the internalstandard for quantification of the amplified nucleic acid.

Suitable amplification methods include, but are not limited to,polymerase chain reaction (PCR) (see Innis et al., PCR Protocols, Aguide to Methods and Application, Academic Press, Inc. San Diego, 1990),ligase chain reaction (LCR) (see Wu and Wallace, Genomics 4:560, 1989;Landegren et al., Science 241:1077, 1988; and Barringer, et al., Gene89:117, 1990), transcription amplification (Kwoh et al., Proc. Natl.Acad. Sci. U.S.A. 86:1173, 1989), and self-sustained sequencereplication (Guatelli et al., Proc. Nat. Acad. Sci. U.S.A. 87:1874,1990). In one embodiment, the sample mRNA is reverse transcribed with areverse transcriptase and a primer consisting of oligo dT and a sequenceencoding the phage T7 promoter to provide single stranded DNA template(termed “first strand”). The second DNA strand is polymerized using aDNA polymerase. After synthesis of double-stranded cDNA, T7 RNApolymerase is added and RNA is transcribed from the cDNA template.Successive rounds of transcription from each single cDNA templateresults in amplified RNA.

Methods of in vitro polymerization are well known to those of skill inthe art (see, for example, Sambrook, supra; Van Gelder et al., Proc.Natl. Acad. Sci. U.S.A. 87:1663-1667, 1990). The direct transcriptionmethod provides an antisense (aRNA) pool. Where antisense RNA is used asthe target nucleic acid, the oligonucleotide probes provided in thearray are chosen to be complementary to subsequences of the antisensenucleic acids. Conversely, where the target nucleic acid pool is a poolof sense nucleic acids, the oligonucleotide probes are selected to becomplementary to subsequences of the sense nucleic acids. Finally, wherethe nucleic acid pool is double stranded, the probes may be of eithersense as the target nucleic acids include both sense and antisensestrands.

In one embodiment, the hybridized nucleic acids are detected bydetecting one or more labels attached to the sample nucleic acids. Thelabels can be incorporated by any of a number of methods. In oneexample, the label is simultaneously incorporated during theamplification step in the preparation of the sample nucleic acids. Thus,for example, polymerase chain reaction (PCR) with labeled primers orlabeled nucleotides will provide a labeled amplification product. In oneembodiment, transcription amplification, as described above, using alabeled nucleotide (such as fluorescein-labeled UTP and/or CTP)incorporates a label into the transcribed nucleic acids. In someembodiments, a multiplex PCR assay is utilized. The assay can be amultiplex assay.

An exemplary assay for assessing expression of gene expression is shownin FIG. 27. In this assay, a first probe specific to a first gene ofinterest is attached to a first detectable bead. Addition probes can beincluded in the assay, such that it is a multiplex assay. Thus, theassay an include a second probe specific to a second gene of interestattached to a second detectable label, a third probe specific to a thirdgene of interest attached to a third detectable label, etc. In someembodiments a single well can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,or 30 probes, each specific to a different gene of interest, and eachattached to a unique detectable label. In this manner, the expression of1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 genes can be evaluate in eachassay. Exemplary probes are shown in FIG. 27B.

Alternatively, a label may be added directly to the original nucleicacid sample (such as mRNA, polyA mRNA, cDNA, etc.) or to theamplification product after the amplification is completed. Means ofattaching labels to nucleic acids are well known to those of skill inthe art and include, for example, nick translation or end-labeling (e.g.with a labeled RNA) by kinasing of the nucleic acid and subsequentattachment (ligation) of a nucleic acid linker joining the samplenucleic acid to a label (e.g., a fluorophore).

Detectable labels suitable for use include any composition detectable byspectroscopic, photochemical, biochemical, immunochemical, electrical,optical or chemical means. Useful labels include biotin for stainingwith labeled streptavidin conjugate, magnetic beads (for exampleDYNABEADS™), fluorescent dyes (for example, fluorescein, Texas red,rhodamine, green fluorescent protein, and the like), radiolabels (forexample, ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P), enzymes (for example, horseradishperoxidase, alkaline phosphatase and others commonly used in an ELISA),and colorimetric labels such as colloidal gold or colored glass orplastic (for example, polystyrene, polypropylene, latex, etc.) beads.Patents teaching the use of such labels include U.S. Pat. Nos.3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and4,366,241.

Means of detecting such labels are also well known. Thus, for example,radiolabels may be detected using photographic film or scintillationcounters, fluorescent markers may be detected using a photodetector todetect emitted light. Enzymatic labels are typically detected byproviding the enzyme with a substrate and detecting the reaction productproduced by the action of the enzyme on the substrate, and colorimetriclabels are detected by simply visualizing the colored label.

The label may be added to the target (sample) nucleic acid(s) prior to,or after, the hybridization. So-called “direct labels” are detectablelabels that are directly attached to or incorporated into the target(sample) nucleic acid prior to hybridization. In contrast, so-called“indirect labels” are joined to the hybrid duplex after hybridization.Often, the indirect label is attached to a binding moiety that has beenattached to the target nucleic acid prior to the hybridization. Thus,for example, the target nucleic acid may be biotinylated before thehybridization. After hybridization, an avidin-conjugated fluorophorewill bind the biotin bearing hybrid duplexes providing a label that iseasily detected (see Laboratory Techniques in Biochemistry and MolecularBiology, Vol. 24: Hybridization With Nucleic Acid Probes, P. Tijssen,ed. Elsevier, N.Y., 1993).

Nucleic acid hybridization simply involves providing a denatured probeand target nucleic acid under conditions where the probe and itscomplementary target can form stable hybrid duplexes throughcomplementary base pairing. The nucleic acids that do not form hybridduplexes are then washed away leaving the hybridized nucleic acids to bedetected, typically through detection of an attached detectable label.It is generally recognized that nucleic acids are denatured byincreasing the temperature or decreasing the salt concentration of thebuffer containing the nucleic acids. Under low stringency conditions(e.g., low temperature and/or high salt) hybrid duplexes (e.g., DNA:DNA,RNA:RNA, or RNA:DNA) will form even where the annealed sequences are notperfectly complementary. Thus, specificity of hybridization is reducedat lower stringency. Conversely, at higher stringency (e.g., highertemperature or lower salt) successful hybridization requires fewermismatches.

One of skill in the art will appreciate that hybridization conditionscan be designed to provide different degrees of stringency. In a oneembodiment, hybridization is performed at low stringency in this case in6×SSPE-T at 37° C. (0.005% Triton X-100) to ensure hybridization andthen subsequent washes are performed at higher stringency (e.g.,1×SSPE-T at 37° C.) to eliminate mismatched hybrid duplexes. Successivewashes may be performed at increasingly higher stringency (e.g., down toas low as 0.25×SSPE-T at 37° C. to 50° C.) until a desired level ofhybridization specificity is obtained. Stringency can also be increasedby addition of agents such as formamide Hybridization specificity may beevaluated by comparison of hybridization to the test probes withhybridization to the various controls that can be present (e.g.,expression level control, normalization control, mismatch controls,etc.).

In general, there is a tradeoff between hybridization specificity(stringency) and signal intensity. Thus, in one embodiment, the wash isperformed at the highest stringency that produces consistent results andthat provides a signal intensity greater than approximately 10% of thebackground intensity. Thus, the hybridized array may be washed atsuccessively higher stringency solutions and read between each wash.Analysis of the data sets thus produced will reveal a wash stringencyabove which the hybridization pattern is not appreciably altered andwhich provides adequate signal for the particular oligonucleotide probesof interest. These steps have been standardized for commerciallyavailable array systems.

Methods for evaluating the hybridization results vary with the nature ofthe specific probe nucleic acids used as well as the controls provided.In one embodiment, simple quantification of the fluorescence intensityfor each probe is determined. This is accomplished simply by measuringprobe signal strength at each location (representing a different probe)on the array (for example, where the label is a fluorescent label,detection of the amount of florescence (intensity) produced by a fixedexcitation illumination at each location on the array). Comparison ofthe absolute intensities of an array hybridized to nucleic acids from a“test” sample (such as from a patient treated with a therapeuticprotocol) with intensities produced by a “control” sample (such as fromthe same patient prior to treatment with the therapeutic protocol)provides a measure of the relative expression of the nucleic acids thathybridize to each of the probes.

Changes in expression detected by these methods for instance can bedifferent for different therapies, for example, and may includeincreases or decreases in the level (amount) or functional activity ofsuch nucleic acids, their expression or translation into protein, or intheir localization or stability. An increase or a decrease can be, forexample, about a 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, change(increase or decrease) in the expression of a particular nucleic acid.

In some examples, the effectiveness of an agent, or the production ofRPE cells, or the identification of RPE cells, is performed by applyingisolated nucleic acid molecules to an array in which the isolatednucleic acid molecules are obtained from a biological sample includingRPE cells, such as following treatment with an agent of interest. Insuch example, the array includes oligonucleotides complementary to thenucleic acids disclosed above.

In an example, the isolated nucleic acid molecules are incubated withthe array including oligonucleotides complementary to nucleic acidmolecules disclosed above for a time sufficient to allow hybridizationbetween the isolated nucleic acid molecules and oligonucleotide probes,thereby forming isolated nucleic acid molecule:oligonucleotidecomplexes. The isolated nucleic acid molecule:oligonucleotide complexesare then analyzed to determine if expression of the isolated nucleicacid molecules is altered. In such example, an agent is evaluated to seeif it affects expression of the molecule as compared to a control (suchcells not contacted with the agent) or reference value.

A gene expression profile is disclosed herein that can be used toidentify the effectiveness of an agent for producing RPE cells, or foridentifying an RPE cell. In an example, the gene expression profileincludes at least two of the molecules listed above, such as at least 5,at least 7, at least 10, at least 20, at least 30, at least 40, or allof the molecules that are listed. In some examples, the gene expressionprofile includes at least 2, at least 5, at least 7, at least 10, atleast 20, at least 25, at least 25, at least 30, at least 35 or at least40 molecules (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 22, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40 or all 41 molecules). In one specificnon-limiting example, the gene expression profile includes of MITF,PAX6, LHX2, TFEC, CDH1, CDH3, CLDN10, CLDN16, CLDN19, BEST1, TIMP3,TRPM1, TRPM3, TTR, VEGFA, CSPG5, DCT, TYRP1, TYR, SILV, SILL MLANA,RAB27A, OCA2, GPR143, GPNMB, MYO6, MYRIP, RPE65, RBP1, RBP4, RDH5,RDH11, RLBP1, MERTK, ALDH1A3, FBLN1, SLC16A1, KCNV2, KCNJ13, and CFTR.

In additional embodiments, the gene expression profile includes at least2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95or all 96 of the miRNAs listed in Table 2. In one specific, non-limitingexample, the gene expression profile includes miR204 and miR211. Infurther embodiments, the gene expression profile includes at least 5,10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100, 105, 110, 115, 120, 125, 130, 135, or all of the markers listed inTable 1 and Table 2. This expression profile can be used to detect RPEcells, and/or confirm that a cell is an RPE cell.

As an alternative to analyzing the sample for the presence of nucleicacids, the presence of proteins can be determined. Proteins can bedetected by any method known in the art. In some examples, proteins arepurified prior to detection. For example, the effect of an agent can bedetermined by incubating the cells, such as iPSCs, embryoid bodies, orRPE cells with one or more antibodies that specifically binds to one ofthe molecules listed above, for an amount of time sufficient to form animmune complex, in order to detect expression. The primary antibody caninclude a detectable label. For example, the primary antibody can bedirectly labeled, or the sample can be subsequently incubated with asecondary antibody that is labeled (for example with a fluorescentlabel). The label can then be detected, for example by microscopy,ELISA, flow cytometry, or spectrophotometry. In another example, thebiological sample is analyzed by Western blotting for the presence orabsence of the specific molecule. In other examples, the biologicalsample is analyzed by mass spectrometry for the presence or absence ofthe specific molecule. In other examples, a first antibody thatspecifically binds a molecule listed above is unlabeled and a secondantibody or other molecule that can specifically binds the firstantibody is labeled. As is well known to one of skill in the art, asecond antibody is chosen that is able to specifically bind the specificspecies and class of the first antibody. For example, if the firstantibody is a human IgG, then the secondary antibody can be ananti-human-IgG. Other molecules that can bind to antibodies include,without limitation, Protein A and Protein G, both of which are availablecommercially. The secondary antibody is incubated with the cells for asufficient time to form an immune complex.

Suitable labels for the antibody or secondary antibody include variousenzymes, prosthetic groups, fluorescent materials, luminescentmaterials, magnetic agents and radioactive materials. Non-limitingexamples of suitable enzymes include horseradish peroxidase, alkalinephosphatase, beta-galactosidase, or acetylcholinesterase. Non-limitingexamples of suitable prosthetic group complexes includestreptavidin/biotin and avidin/biotin. Non-limiting examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin. A non-limiting exemplary luminescent materialis luminol; a non-limiting exemplary magnetic agent is gadolinium, andnon-limiting exemplary radioactive labels include ¹²⁵I, ¹³¹I, ³⁵S or ³H.

In an alternative example, proteins can be assayed in a biologicalsample by a competition immunoassay utilizing protein standards(molecules) labeled with a detectable substance and unlabeled antibodythat specifically bind to the desired molecule. In this assay, thecells, the labeled molecule standard and the antibody that specificallybinds to the molecule are combined and the amount of labeled moleculestandard bound to the unlabeled antibody is determined. The amount ofthe molecule in the biological sample is inversely proportional to theamount of labeled molecule standard bound to the antibody thatspecifically binds the molecule.

In yet other embodiments the resting potential of the cells is assessed.In some embodiments, a resting potential of about −50 to about −60 mV,and a fluid transport rate of about 5 to about 10 μl cm⁻²h⁻¹ indicatesthat the cells is an RPE cell. In additional embodiments, the productionof cells that express MITF, PAX6, LHX2, TFEC, CDH1, CDH3, CLDN10,CLDN16, CLDN19, BEST1, TIMP3, TRPM1, TRPM3, TTR, VEGFA, CSPG5, DCT,TYRP1, TYR, SILV, SILL MLANA, RAB27A, OCA2, GPR143, GPNMB, MYO6, MYRIP,RPE65, RBP1, RBP4, RDH5, RDH11, RLBP1, MERTK, ALDH1A3, FBLN1, SLC16A1,KCNV2, KCNJ13, and CFTR, express miR204 and miR211, and have a restingpotential of about −50 to about −60 mV. In certain embodiments, anincrease in the number of cells with these features, following contactwith the agent of interest, indicates that the agent increases theproliferation and/or differentiation of RPE cells.

In additional embodiments, the fluid transport of the cells isdetermined. Fluid transport of cells can be measured using a capacitanceprobe. In some embodiments, cells grown as a monolayer tissue aremounted in a modified Üssings chamber and probe that measure capacitanceis used. If the volume of fluid on either side of the cell layerchanges, capacitance of the probes changes. This can be used tocalculated the volume change and the fluid flow. In some examples, afluid transport rate of about 5 to about 15 μl cm⁻²h⁻¹ such as a fluidtransport rate of about 5 to about 10 μl cm⁻²h⁻¹, indicates that thecell is an RPE cell.

The ability of cells to reversibly increase fluid flow by either ATPtreatment of the apical side or IFN gamma treatment of the basal sidecan be measured. Both of these responses can be inhibited by inhibitingchloride channels on the basal side. These properties can be used todetermine that the cell as an RPE cell.

In further examples, the polarization of the cell is assessed. Thus, insome embodiments, selective CO₂ permeability of the apical surface ofRPE cells is measured. Photoreceptors secrete high concentrations of CO₂towards the apical surface (Adijanto et al., J Gen Physiol. 2009 June;133(6):603-22). Therefore, this surface has been evolutionarily selectedto trap CO₂. This function can be measured in vitro by changing CO₂concentrations in the apical or the basal baths of RPE cells, such asRPE cells growing in transwells. If the RPE cells trap CO₂, they respondby a reduction in pH, which can be measured by a ratio-metric dye.

In one specific non-limiting example, a RPE monolayer grown on a porouspolyester membrane is incubated in Ringer solution containing 8 μMBCECF-AM. After incubation with BCECF-AM, the tissue was incubated incontrol (5% CO₂) Ringer for another 30 minutes before mounting in amodified Üssing chamber. The Üssing chamber is mounted on the stage ofan axiovert-200 microscope (Carl Zeiss, Inc.) equipped with a 20×objective. The RPE is continuously perfused with Ringer solution(equilibrated with 5% CO₂ at 36.5° C.). Excitation photons (440/480 nm)are generated by a xenon light source, and the specific wavelengths areselected with a monochromator (Polychrome IV; Photonics). The emissionfluorescence signals are captured with a photomultiplier tube (ThornEMI). pHi calibrations are performed by perfusing high-K calibrationsolutions (at pH 6.8, 7.2, and 7.6) containing 20 μM nigericin into bothsolution baths.

In some embodiments, changes in the electric properties of RPE cells canbe measured. In some specific non-limiting examples, RPE cells are grownas a monolayer on a porous polyester membrane. Transepithelial potentialis measured with a pair of calomel electrodes in series with Ringersolution agar (4% wt/vol) bridges placed in the apical and basal bathsof the Üssing chamber. The TEP recordings are moving averages of 3seconds. The transepithelial resistance was calculated from Ohm's law.

$R_{T} = {\frac{\Delta\;{{TEP} \cdot {Area}}}{Current}.}$When light hits the photoreceptors in RPE cells, the photoreceptorsdepolarize by closing potassium (K) channels (see, Millers et al., (ed).Encyclopedia of the eye, ELSEVIER). This reduces the concentration ofK-ions in between photoreceptors and RPE (subretinal space) from 5 mM to1 mM. RPE cells respond to this changing concentration byhyperpolarizing and opening their K channels to increase the subretinalK concentration back to 5 Mm. A range of 1-5 mM indicates the cell is anRPE cell.

In further embodiments, the production of cells that express MITF, PAX6,LHX2, TFEC, CDH1, CDH3, CLDN10, CLDN16, CLDN19, BEST1, TIMP3, TRPM1,TRPM3, TTR, VEGFA, CSPG5, DCT, TYRP1, TYR, SILV, SILL MLANA, RAB27A,OCA2, GPR143, GPNMB, MYO6, MYRIP, RPE65, RBP1, RBP4, RDH5, RDH11, RLBP1,MERTK, ALDH1A3, FBLN1, SLC16A1, KCNV2, KCNJ13, and CFTR, express miR204and miR211, have a resting potential of about −50 to about −60 mV, andhave a fluid transport rate of about 5 to about 10 μl cm⁻²h⁻¹ i. Incertain embodiments, an increase in the number of cells with thesefeatures, following contact with the agent of interest, indicates thatthe agent increases the proliferation and/or differentiation of RPEcells.

The disclosure is illustrated by the following non-limiting Examples.

EXAMPLES Example 1 Exemplary Materials and Methods for Differentiationof Human Induced Pluripotent Stem Cells (hiPSC) into Retinal PigmentEpithelial Cells (RPE)

Maintaining and Passaging hiPSC

Grow hiPSC on g-irradiated mouse embryo fibroblasts (MEFs) in humanembryonic stem (hES) media+basic Fibroblast Growth Factor (bFGF-addedfresh).

Feed hiPSC daily with hES media+bFGF (added fresh) to a finalconcentration of 10 ng/ml-1.5 ml/well of P6.

When hiPSC colonies are 80% confluent, split them (1:6) onto new MEFsfor continued passage (eg. six wells of a P6 plate expanded into six-P6plates)

Aspirate hES media

Wash 1× with PBS

Add 1 ml CTK and place in +37 C incubator for 10 min (time varies withiPSC line)

Aspirate CTK (make sure colonies are still adhered to the plate)

Add 2 ml PBS without Ca/Mg and place in +37 C incubator for 5 min

Aspirate PBS and add another 2 ml of PBS and place in +37 C incubatorfor 5 min

Aspirate PBS (MEFs are preferentially lifted in the PBS)

Add 1 ml of hES media+(10 ng/ml) bFGF (added fresh)+(10 uM) RockInhibitor (RI) (added fresh) to each well of P6

Using a 1 ml pipetman, gently pipet up and down to remove colonies fromplate (try not to generate single cells); transfer cells to a cleantube; add another 1 ml hES media+bFGF+RI to each well, turn plate, andpipet up and down to loosen remaining colonies; transfer cells to sametube; add the appropriate volume of hES+bFGF+RI media to cells (6-P6plates will require 54 mls)

Aspirate Feeder media from MEF plates

Distribute 1.5 mls cells/well of P6

(Reh et al., 2010, Methods Mol. Biol.; Takahashi et al., 2001 PLoS One)

When hiPSC colonies are 70% confluent, prepare embryoid bodies (EBs)

Aspirate hES media

Wash 1× with PBS

Add 1 ml of 1 mg/ml collagenase/well of P6 and put in +37 C incubatorfor 15 min

Aspirate collagenase and add 1 ml DMEM

Scrape colonies gently with cell lifter (Corning#3008) being careful notto break the colonies into single cells

Transfer contents of all 6 wells to a 15 ml conical tube with a 1 mlpipetman

Add another 1 ml of DMEM to each well, turn the plate around, and gentlyscrape the remaining colonies with the cell lifter; transfer contents ofall 6 wells to the same tube (˜12 ml DMEM/tube).

Let the cell aggregates settle to the bottom of the tube (˜2-5 min oruntil the media is clear).

Aspirate the supernatant (contains the single cell and MEFs) and washthe cell aggregate with 10 ml DMEM; pipet up and down with 10 ml pipet3-5 times to break up aggregates-if they are too big (will depend onsize of starting iPSC colony); break them into smaller clumps. Smallercell clumps that contain between 200-500 cells make RPE with a higherefficiency, as compared to larger clumps. Larger clumps when plated make3-dimensional structures that do not form RPE at a high efficiency.

Let the cell aggregates settle to the bottom of the 15 ml conical tube.

Aspirate the supernatant and resuspend the cell aggregate in desiredvolume of Retinal Induction Medium (RIM)+Rock inhibitor (10 uM) (usually8 ml of RIM/15 ml conical tube). Reduction of the amount of knockoutserum replacement (KSR) from 10% to 1.5% increased RPE differentiationefficiency.

Transfer aggregates in RIM+RI to a low attachment dish (Corning#326210cm low attachment plate) where they will form EBs within 24 hours

48 hours later, evenly distribute EBs onto plates coated with MATRIGEL®.150-250 EBs/well of a 6-well plate differentiate into RPE with a higherefficiency as compared to more than 250 EBs/well. Higher number of EBsmakes too confluent cultures and cells do not differentiate into RPE ata high efficiency.

Transfer EBs to 15 ml conical tube and let settle (˜2-5 min).

Aspirate supernatant.

Add appropriate volume of Retinal Differentiation Media (RDM) to EBs.

Aspirate the matrigel from each well and wash 1× with PBS.

Distribute equal amount of EBs to each matrigel-coated well and swirlplate to achieve an even distribution of EBs on the well's surface(usually EBs from 1-well of a P6-plate can be plated into 2-wells of aP6-plate).

Change media every other day for 3 weeks (eg. Mon, Wed, Fri)

This protocol significantly increased the expression of genesresponsible for RPE fate induction and have increased the efficiency ofRPE differentiation. The elements of this protocol are a reduced conc.of KSR (1.5%), no FGF, addition of DKK1 (100 ng/ml), and PD 0325901 (0.1uM), using NOGGIN at 50 ng/ml. Inhibition of both WNT (by DKK1) and BMP(by NOGGIN) pathways promotes rostral neuroectoderm formation, which canalso make eye cells. Inhibition of FGF signaling (by PD 0325901)suppressed retinal fate and induces RPE fate from rostralizedneuroectoderm.

Transfer cells to Retinal Media (RM) plus Nicotinamide, Activin, andWnt3a (NAW). Change media every other day (eg. Mon, Wed, and Fri) for 3weeks.

Addition of WNT3a has also significantly increased the RPEdifferentiation efficiency. Activation of canonical WNT signalingincreases the expression of MITF and PAX6, two RPE inducingtranscription factors and thus increases RPE differentiation efficiency.

Transfer cells to 5% Retinal Pigmented Epithelial (RPE) Media containingWNT5a (100 ng/ml), DKK1 (100 ng/ml), SU5402 (10 uM), CYCLOPAMINE (5 uM).WNT5a and DKK1 inhibit canonical WNT signaling, thereby reducing PAX6and MITF expression and maturing RPE cells. SU5402 inhibits FGFsignaling and CYCLOPAMINE inhibits Sonic signaling. Both FGF and Sonicsignaling are associated with retinal induction. Therefore, inhibitingthese pathways promotes RPE differentiation. Change media every otherday (for example: Monday, Wednesday, and Friday). Within a week or twoseveral pigmented colonies appear. At this point, cells can be seededonto transwells in 5% RPE medium for functional analysis.

Cells are maintained for 8 weeks onto these transwells, where theycontinue to mature. At 8 weeks fully functional and polarized monolayersof cells on transwells are harvested for the expression of RPE-signaturegenes and miRNAs. Gene expression is measured using qRT-PCR assays.

To Prepare MEF Plates:

Coat 4-P6 plates with 0.1% gelatin and let sit at room temp for at least15 min

Thaw 1 vial of MEFs (Mt. Sinai Stem Cell #GSC-6001G; 5 millioncells/vial) and resuspend in 9 ml Feeder Cell Media

Spin cells 1.2K for 5 min; aspirate supernatant and resuspend pellet in36 ml Feeder Cell Media

Aspirate gelatin from wells and distribute 1.5 ml MEFs/well of P6

-   -   MEFs are good for 2 weeks and do not have to change media        To Prepare MATRIGEL® Plates:

Let matrigel (BD #356230) thaw overnight on ice at +4 C.

Add equal volume of DMEM to thawed matrigel on ice the next day and mixwell.

Incubate overnight on fresh ice at +4 C.

Mix the next day and aliquot 0.4 ml/eppendorf tube (=1:2) and freeze at−80 C.

Thaw a tube of diluted (1:2) matrigel on ice (or let it thaw over-nightat +4 C); keep the aliquots on ice while working with them.

Dilute the matrigel 1:25 in cold DMEM media (10 ml media+0.4 mlMATRIGEL® (1:2)).

To coat a tissue culture plate, place matrigel on the dish and shake todistribute evenly (for example 1 ml/well of a P6).

Let the plate sit at room temperature for 1 h to overnight with verygentle shaking; chill the plates at +4 C before use.

If the plates are not used immediately, wrap them in foil and keep at +4C for 1-2 days.

Reagents

Feeder Cell Media (500 ml)

DMEM (High Glucose) (Invitrogen#11960-077) 435 ml  Heat-InactivatedFetal Bovine Serum (FBS) 50 ml  (Invitrogen#10082147) Pen/Strep(Gibco#15140) 5 ml Glutamine (200 mM) (Gibco#25030) 5 ml Na Pyruvate(100 mM) (Gibco#11360) 5 mlFilter MediahES Media (500 ml)

DMEM/F12 (1:1) (Invitrogen#11320-033) 385 ml Knockout Serum Replacement(Invitrogen 100 ml #10828-028) Non-Essential Amino Acids (Gibco#11140) 5 ml Pen/Strep (Gibco#15140)  5 ml Glutamine (200 mM) (Gibco#25030)  5ml b-Mercaptoethanol (55 mM in PBS) (Gibco#21985)  0.5 mlFilter Media.

Add bFGF (RD Systems#233-FB; final concentration of 10 ng/ml; make 10ug/ml stock and dilute 1:1000 in hES) to hES media immediately beforeuse. When passaging hiPSC, also add RI to hES media immediately beforeuse.

CTK:

5 ml 0.25% Trypsin+5 ml 1 mg/ml collagenase IV+0.5 ml 0.1M CaCl2+10 mlKSR (knockout serum)+30 ml DMEM-F12, store @-20 C

Rock inhibitor from EMD In solution Y27632 cat #688001—10 mM stocksolution

Retinal Induction Medium (RIM) (500 ml)

DMEM/F12 (1:1) (Invitrogen#11320-033) 500 ml Knockout Serum Replacement(Invitrogen #10828-028) 7.5 ml Non-Essential Amino Acids (Gibco#11140) 5ml Pen/Strep (Gibco#15140) 5 ml Sodium Pyruvate (100 mM) (Gibco#11360) 5ml N2 Supplement 1x (Gibco#17502-048) 5 ml B27 Supplement 1x(Gibco#17504-044) 10 ml CK1-7 Dihydrochloride (5 mM) (Sigma#C0742) 50 ulSB 431542 hydrate (5 mM) (Sigma#S4317) 50 ul Noggin (250 ug/ml) (RDSystems#6057-NG) 2 ul IGF-1 (100 ug/ml) (RD Systems#291-G1) 5 ulAscorbic Acid (5 mg/ml) (Sigma#A4544-25G) 5 mlRetinal Differentiation Medium (RDM) (500 ml)

DMEM/F12 (1:1) (Invitrogen#11320-033) 500 ml Knockout Serum Replacement(Invitrogen #10828-028) 7.5 ml Non-Essential Amino Acids (Gibco#11140) 5ml Pen/Strep (Gibco#15140) 5 ml Sodium Pyruvate (100 mM) (Gibco#11360) 5ml N2 Supplement 1x (Gibco#17502-048) 5 ml B27 Supplement 1x(Gibco#17504-044) 10 ml CK1-7 (5 mM) (Sigma#C0742) 0.5 ml DKK1 (100ug/ml) (R&D Systems) 0.5 ml SB-431542 (5 mM) (Sigma#S4317) 0.5 ml Noggin(250 ug/ml) (RD Systems#6057-NG) 0.1 ml IGF-1(100 ug/ml) (RDSystems#291-G1) 50 ul Ascorbic Acid (5 mg/ml) (Sigma#A4544-25G) 5 ml PD0325901 (1 mM) (Tocris#4192) 0.5 ml NO FGFRetinal Medium (RM)+Nicotinamide, Activin, and Wnt3a (NAW) (500 ml)

DMEM/F12 (1:1) (Invitrogen#11320-033) 500 ml Knock out Serum Replacement(Invitrogen #10828-028) 50 ml Non-Essential Amino Acids (Gibco#11140) 5ml Pen/Strep (Gibco#15140) 5 ml Sodium Pyruvate (100 mM) (Gibco#11360) 5ml N2 Supplement 1x (Gibco#17502-048) 5 ml B27 Supplement 1x(Gibco#17504-044) 10 ml Nicotinamide (Sigma#N0636) (1M) 5 ml Activin (RDSystems#338-AC) (50 ug/ml) 1.5 ml Wnt3a (RD Systems#5036-WN) (200 ug/ml)250 ul

Rock inhibitor from EMD In solution Y27632 cat #688001

5% Retinal Pigmented Epithelial (RPE) Media

MEM_modified (M-4526) 500 ml N1 supplement (N-6530) 5 ml Glutamax,Penicillin-streptomycin (G-1146) 5 ml Non essential amino acids (M-7145)5 ml Taurine (T-0625) 125 mg Hydrocortisone (H-0396) 10 ugTriiodo-thyronin (T-5516) 0.0065 ug Fetal bovine serum, heat inactivated1% or 5% or 15%

The use of different amounts of Knockout Serum Replacement (KOSR) in themedia used for the first three weeks of the differentiation protocol wasassessed. Retinal differentiation media (RDM), showed differentialeffects on cell pigmentation. iPSC were incubated in high KOSR (10%)resulted in a smaller number of pigmented cells compared to the use oflesser amounts of KOSR (1.5%). After culture in 10, 5.25, or 1.5% KOSR,the cells were shifted to NAW containing 1.5% KOSR for 3 weeks andphotographed at 16 weeks (see FIG. 34A and FIG. 34B).

Example 2 Reporter iPSC Cell Lines

To optimize the protocol for iPSC to RPE differentiation, a reporteriPSC line was created. This reporter line constitutively expresses a redfluorescent protein, whose expression is controlled by ubiquitin genepromoter. The reporter iPSC line also expresses a green fluorescentprotein (GFP), specifically when differentiated in to RPE lineage (FIG.1-3). The RPE-specific enhancer of the gene Tyrosinase controls GFPexpression in this iPSC line (FIG. 1). The first goal was to confirmthat the GFP expression truly represents RPE lineage. To achieve this,iPSC line were differentiated using published differentiation protocolsand characterized those differentiated cells using molecular andphysiological assays. These assays are disclosed in FIGS. 22-24. FIG. 2describes a simple schematic drawing of iPSC-RPE differentiation. Whenthe reporter iPSC were differentiated into RPE, approximately 28 daysinto differentiation, GFP positive cells are present (FIG. 3A). Thesecells go on to form an epithelial monolayer with characteristicpolygonal RPE morphology and continue to express GFP (FIG. 3B). GFPpositive and negative cells were sorted by flow cytometry and theexpression of several RPE-specific genes was analyzed, shown in FIG. 4.The results show that GFP positive cells express RPE-specific markers atmuch higher levels as compared to GFP negative cells from the same dish.

Next, sorted GFP positive and negative cells were re-plated onto newdishes. GFP negative cells grow to form very few pigmented colonies(FIG. 5A). In comparison, GFP positive cells develop in to pure, fullyconfluent RPE monolayers (FIG. 5B). This confirmed that the GFPexpression truly represents RPE-fate in these cells and demonstratedthat GFP signal can be used as a reliable indicator of RPEdifferentiation. To optimize differentiation of RPE from iPSCs, areference protocol was established (see Idelson et al. (2009), Cell StemCell 5, 396-408; Meyer et al. (2009), Proc. Natl. Acad. Sci. USA 106,16698-16703; Osakada et al. (2009), J. Cell Sci. 122, 3169-3179. Theprotocol was divided into four stages—induction of differentiation,differentiation into eye-field/optic vesicle cells, induction of RPEdifferentiation, and maturation of RPE cells.

Example 3 Induction of Differentiation

To induce differentiation of iPSCs, the colonies were lifted by a mildcollagenase treatment (1 mg/ml for 15 minutes) and dissociated intosmall cell aggregates. These aggregates were cultured in RIM medium for48 hours in non-adherent culture conditions. Within 24 hours, the cellaggregates change into embryoid bodies (EBs). It was determined whetherthe size of EBs (number of cells/EB) affects RPE differentiation andfound that relatively smaller EBS with 200-400 cells/EB efficientlydifferentiate into RPE as compared to larger EBs (FIG. 6).

Retinal Induction Medium (RIM) (500 ml)

DMEM/F12 (1:1) (Invitrogen#11320-033) 500 ml Knockout Serum Replacement(Invitrogen #10828-028) 7.5 ml Non-Essential Amino Acids (Gibco#11140) 5ml Pen/Strep (Gibco#15140) 5 ml Sodium Pyruvate (100 mM) (Gibco#11360) 5ml N2 Supplement 1x (Gibco#17502-048) 5 ml B27 Supplement 1x(Gibco#17504-044) 10 ml CK1-7 Dihydrochloride (5 mM) (Sigma#C0742) 50 ulSB 431542 hydrate (5 mM) (Sigma#S4317) 50 ul Noggin (250 ug/ml) (RDSystems#6057-NG) 2 ul IGF-1 (100 ug/ml) (RD Systems#291-G1) 5 ulAscorbic Acid (5 mg/ml) (Sigma#A4544-25G) 5 ml

Example 4 Differentiation into Eye-Field/Optic Vesicle Cells

After 48 hours of floating EB cultures they are plated onto MATRIGEL®coated plates in RDM medium. It was investigated whether the number ofEBs plated/well affects differentiation of iPSCs into RPE. The resultsshowed that ideal differentiation efficiency is achieved by plating150-250 EBs/well of a standard 6-well tissue culture plate (FIG. 7). RDMmedium was used for 3 weeks with change of medium on cells every Monday,Wednesday, and Friday. Inhibition of Nodal, BMP, and WNT pathways can beimportant for differentiation of stem cells into eye-field cells. Toinhibit the nodal pathway, the inhibitor SB431542 was used. To inhibitthe activity of BMP pathway, various concentrations of Noggin, a BMPinhibitor, were tested. For WNT pathway inhibition, a combination ofbiological Wnt inhibitor DKK1 and a small molecule inhibitor of the WNTpathway CK1-7 were used.

Results have shown that inhibition of FGF signaling is important for RPEdevelopment. Therefore, the activity of FGF signaling pathway wasmodulated by either changing its concentration or/and by adding FGFinhibitor (PD 0325901) to the medium. The results were scored weekly byquantitative gene expression measurements of two transcription factorsPax6 and Mitf which are the master regulators of RPE differentiation(FIGS. 8-13). In addition, GFP expression was scored after six weeks asa direct measure of induction of RPE cells (FIG. 14).

The results showed that dual inhibition of WNT pathway, inhibition ofFGF pathway, and use of 50 ng/ml of Noggin significantly andprogressively improve the expression of Pax6 and Mitf during the threeweeks, suggesting that the cells have attained optic vesicle identity bythe end of the third week. The GFP signal measured at the end of normalRPE differentiation confirms that these treatments significantly improveRPE differentiation from iPSCs.

Next, the effect of the concentration of knockout serum replacement(KSR) on RPE differentiation was tested. Reducing KSR to 1.5% increasedthe number of pigmented RPE cells when differentiated from the RPE (FIG.15).

Retinal Differentiation Medium (RDM) (500 ml)

DMEM/F12 (1:1) (Invitrogen#11320-033) 500 ml Knockout Serum Replacement(Invitrogen #10828-028) 7.5 ml Non-Essential Amino Acids (Gibco#11140) 5ml Pen/Strep (Gibco#15140) 5 ml Sodium Pyruvate (100 mM) (Gibco#11360) 5ml N2 Supplement 1x (Gibco#17502-048) 5 ml B27 Supplement 1x(Gibco#17504-044) 10 ml CK1-7 (5 mM) (Sigma#C0742) 0.5 ml DKK1 (100ug/ml) (R&D Systems) 0.5 ml SB-431542 (5 mM) (Sigma#S4317) 0.5 ml Noggin(250 ug/ml) (RD Systems#6057-NG) 0.1 ml IGF-1 (100 ug/ml) (RDSystems#291-G1) 50 ul Ascorbic Acid (5 mg/ml) (Sigma#A4544-25G) 5 ml PD0325901 (1 mM) (Tocris#4192) 0.5 ml NO FGF

Example 5 Induction of RPE Differentiation

Activin A induces RPE differentiation from ES cells. In animal models,canonical WNT (WNT3a) is important for RPE differentiation. It washypothesized that a combination of activin A and WNT3a would beeffective for the induction of RPE differentiation from iPSCs. Acombination of these two factors was tested at the optic vesicle stage.Cells were moved from the RDM medium to medium containing either activinA (NA) only or activin A plus WNT3a (NAW). The addition of WNT3asignificantly increased the number of pigmented cells per dish (FIG.16). Because this differentiation process was started with the reporteriPSC lines, the increase in the number of RPE cell was confirmed bycomparing the GFP signal between the two treatments. As compared to NA,NAW increased the number of GFP positive cells in the culture (FIG. 17,top panel). When quantified by FACS analysis, NA treatment produced only22% GFP positive cells, whereas NAW treatment produced 52% GFP positivecells (FIG. 17, bottom panel). This result showed that WNT signalingsynergistically enhances RPE differentiation capacity of TGF-beta(activin) signaling.

Retinal Medium (RM)+Nicotinamide, Activin, and Wnt3a (NAW) (500 ml)

DMEM/F12 (1:1) (Invitrogen#11320-033) 500 ml Knock out Serum Replacement(Invitrogen #10828-028) 7.5 ml Non-Essential Amino Acids (Gibco#11140) 5ml Pen/Strep (Gibco#15140) 5 ml Sodium Pyruvate (100 mM) (Gibco#11360) 5ml N2 Supplement 1x (Gibco#17502-048) 5 ml B27 Supplement 1x(Gibco#17504-044) 10 ml Nicotinamide (Sigma#N0636) (1M) 5 ml Activin (RDSystems#338-AC) (50 ug/ml) 1.5 ml Wnt3a (RD Systems#5036-WN) (200 ug/ml)250 ul

Example 6 Maturation of RPE Cells

To further purify and mature RPE cells, these cells were trypsinized andcultured in 5% RPE medium for 4 weeks on semipermeable transwells. Thisproduced cells which looked quite homogenous in terms of RPE pigment(FIG. 18, top panel). Quantification of the GFP signal by FACS analysisproduced up to 92% GFP positive cells in cultures that have grown for 4weeks. This suggests that the 5% RPE medium enriches for RPE cells. Whenthese cells were cultured for longer durations, NAW treated cells lookedmore homogenous in terms of RPE pigment, RPE morphology, and GFPexpression as compared to NA treated cells (FIG. 19, FIG. 20 only forNAW cells). However, these cells continue to express fetal specific RPEgenes such as Pax6, Sox2, Tfec etc.

Differentiating RPE cells start producing canonical WNT that can affectthe maturity of RPE cells. Therefore, to generate RPE cells that arefully mature and stop expressing fetal genes, these cultures weretreated with inhibitors of canonical WNT pathway. WNT5a and DKK1, whichtogether inhibit canonical WNT pathway and induce non-canonical WNTpathway, were used in these studies. The later has been shown to inducecell polarization and maturity. In addition, inhibitors of FGF (SU54052)and Sonic hedgehog (Cyclopamine) pathways were used to suppress anyendogenous FGF and Sonic signaling, which may affect RPE maturity. Theseinhibitors were used for 2 weeks and the cells were assayed immediatelyafter the treatment. The expression of several fetal RPE and mature RPEspecific genes were measured (FIG. 21). Interestingly, these treatmentsdown-regulated the expression of fetal RPE specific genes (Pax6, Sox2,Tfec, Klf4, Snail1/2) and up-regulated the expression of RPE maturityassociated genes (TYR, TYRP1, MYRIP, Cadherin 1/3, TRPM1/3).

In conclusion, a protocol was developed that significantly improves iPSCto RPE differentiation and also generates cells that are fullydifferentiated. This protocol is extremely valuable to produce RPE cellsfrom iPSCs that can serve appropriate disease models and an effectivecell-based therapy.

5% Retinal Pigmented Epithelial (RPE) Media

MEM_modified (M-4526) 500 ml N1 supplement (N-6530) 5 ml Glutamax,Penicillin-streptomycin (G-1146) 5 ml Non essential amino acids (M-7145)5 ml Taurine (T-0625) 125 mg Hydrocortisone (H-0396) 10 ugTriiodo-thyronin (T-5516) 0.0065 ug Fetal bovine serum, heat inactivated5% DKK1 (100 ug/ml) (R&D Systems) 0.5 ml Wnt5a (RD Systems) (100 ug/ml)250 ul SU5402 (sigma) (10 uM) 0.5 ml Cyclopamine (Sigma) (10 uM) 0.5 ml

Example 7 Authentication of iPSC-Derived RPE Cells

To authenticate the cells obtained through this protocol, the cells weretested by both molecular and physiological assays. A set of signaturegenes was selected from a previously published RPE-gene signature(Strunnikova et al. (2010), Hum. Mol. Genet. 19: 2468-2486) and theexpression of those genes was compared between primary human RPE and RPEobtained from iPSCs using our protocol. The resulting cells closelyresemble the primary human RPE in terms of the expression ofRPE-specific genes (FIG. 22).

Similarly, the ability of iPSC-derived RPE to respond to changing CO₂concentrations in the apical and basal baths, and their ability tohyperpolarize in response to changing potassium concentrations in theapical bath was assessed (FIG. 23, 24). In all these assays,iPSC-derived RPE cells behave similar to primary human RPE. Thus, thedisclosed protocol produces RPE cells that are fully functional. Thesemolecular and physiological assays are important biomarkers for completeRPE differentiation.

Example 8 Multiplex High Throughput Screening

A multiplex high-throughput screening approach was developed thatsimultaneously detects endogenous expression of multiple developmental,functional, and disease markers in iPSC-derived retinal pigmentepithelium (RPE). Protocols were optimized for differentiation andgrowth of iPSC-derived RPE for high-throughput screening in 96-well and384-well formats. As a proof of principle, the endogenous expression often genes in iPSCs, iPSC-derived RPE at two differentiation stages, andprimary human RPE cells were compared using the multiplex assay. Dataobtained from the assay are significantly correlated with standardqRT-PCR based measurements. This assay provides the basis to screen forcompounds that improve RPE function, maturation, and target diseasepathways thus providing the basis for effective treatments of severalretinal degenerative diseases.

Protocols were developed to use fully-authenticated iPSC-derived RPE fora multiplex high-throughput assay. This multiplex gene expression assayreports on six RPE lineage genes, two stem/progenitor cell genes, andtwo house-keeping genes. Proof of principle data was obtained that 1)the assay can be performed in 96-well and 384-well high-throughputmodes, 2) the assay is able to measure even subtle change in geneexpression, 3) the data obtained with the multiplex assay is highlycorrelated with RT-qPCR data. This assay allows identification of smallmolecules that can further enhance the efficiency of the differentiationprotocols toward fully mature RPE cells. In addition, it providesdevelopmental and functional biomarkers that can be tracked in ahigh-throughput mode. Small molecules that modulate the expression ofthese functional and disease biomarkers can provide potentialtherapeutic drugs for RPE-associated retinal degenerative diseases.

A. Materials and Methods

iPSC Derivation, Culture, and Differentiation:

Human adult dermal fibroblasts (AG9309, female, 21 years old, toebiopsy) purchased from Coriell (Camden, N.J.) were reprogrammed (15).iPSC were differentiated into three germ layers or into RPE (12, 15-17).

Immunostaining, Electron Microscopy, and qRT-PCR Analysis:

Immunostaining and qRT-PCR analysis was performed (Green et al., Naturebiotechnology 29(3):267-272, 2011; Bharti et al., PLoS Genet8(7):e1002757, 2012). Following antibodies were used: ALEXAFLUOR®488-anti Tra-1-60 (1:50, BD Biosciences); ALEXAFLUOR® 488—anti SSEA4(1:50, BD Biosciences); OCT4 (1:400, Cell Signaling, Danvers, Mass.);NANOG (1:100, R&D Systems); SOX2 (1:100, Santa Cruz, Dallas, Tex.); KLF4(1:50, Santa Cruz); c-MYC (1:50, Santa Cruz); AFP (1:75, ThermoScientific, Waltham, Mass.); TUJ1 (1:400, Sigma, St. Louis, Mo.,http://www.sigmaaldrich.com); aSMA (1:500, Thermo Scientific); DCT(1:100, Bioworld antibodies, Mt. Airy); PAX6 (1:200, Covance Chantilly,Va.); and ZO1 (1:100 Life Technologies).

Intracellular Calcium Measurements and Electrophysiology:

To assess calcium signaling in the RPE cells: ATP (200 μM; Sigma) andCyclopiazonic Acid (CPA; 10 μM; EMD Millipore); (see Maminishkis et al.,Invest. Ophthal. Vis. Sci. 47(8):3612-3624, 2006); Quinn et al., Invest.Ophthal. Vis. Sci. 33(13):3513-3527, 1992).

Calomel electrodes in series with Ringer solutions and agar bridges wereused to measure the transepithelial potential (TEP). The signals fromintracellular microelectrodes were referenced to the basal bath tomeasure the apical and basal membrane potentials, V_(A) and V_(B), whereTEP=V_(B)−V_(A). The total transepithelial resistance (R_(t)), and theratio of the apical to basolateral membrane resistance (R_(A)/R_(B))were obtained by passing 2-4 μA current pulses (peak to peak) across thetissue and measuring the resultant changes in TEP, V_(A) and V_(B). SeeMaminishkis et al. supra and Quinn et al., supra for details.

Principle of QUANTIGENE PLEX® Technology:

Cells grown in 96-well or 384-well plates are lysed and the lysatetransferred to a hybridization plate containing QUANTIGENE PLEX® probeand LUMINEX® beads sets. Each bead type is coated with a differentsingle strand DNA Capture Probe (CP). Other components of theQUANTIGENE® Probe set that are also comprised of ssDNA oligos includeCapture Extenders (CE), Label Extenders (LE) and Blocking Probes (BP).Part of CE oligos is complementary to the target mRNA (cover 200-600bases) and part to CP bound on LUMINEX® beads. This interactionfacilitates capture of specific target mRNAs to specific LUMINEX® beads.LE include target mRNA specific sequences and a binding site for thepreamplifier, the first component used for signal amplification. BP bindto any sequences on the target mRNA that are not bound by the CE and LE.A typical probe set for a single target mRNA consists of a family offour or more different CE and LE that usually cover about 500 baseswithin the target mRNA. After washing off excess probes and remainingcell lysate the signal amplification reagents consisting of thePreamplifier (PreAmp), Amplifiers (Amp), and Label Probes (LP) aresequentially hybridized to the mRNAs. The LP also includes a biotinmolecule, which in turn is a binding site for the final signalamplification reagent, SAPE (Streptavidin-conjugated R-Phycoerythrin).Each Amplifier binds up to 400 SAPE. The resulting fluorescence signalassociated with individual Capture Beads is read on a LUMINEX®instrument. Signal is reported as median fluorescence intensity (MFI)and is proportional to the number of target RNA molecules present in thesample. MFI is calculated by measuring signals on 50-100 beads/gene. MFIvalues obtained from background blank wells with no target RNA weresubtracted from MFI of each target reading.

QUANTIGENE PLEX® 2.0 Reagent System:

96-well Assay Protocol: The QUANTIGENE PLEX® 96-well Assay was done asdescribed in the manufacturer's manual (QUANTIGENE PLEX® 2.0 Assay,Affymetrix, Santa Clara, Calif.). 25,000 and 50,000 cells are seeded perwell and cultured for 14 days at 37° C., 5% CO₂. Cells were lysed in 200μL working lysis mixture for 30 minutes at 50° C. 80 μL of cell lysatewere transferred to the assay's hybridization plate (96-well clearpolypropylene plate Abgene #AB0796, Pittsburgh, Pa.), where each wellalready contained 20 μL Working Bead Mix (6.6 μL Lysis Mixture, 5.2 μLnuclease free water, 0.2 μL Proteinase K solution, 2 μL BlockingReagent, 5 μL Probe Set, 1 μL magnetic LUMINEX® beads; QUANTIGENE PLEX®Set-Panel #11828). Hybridization was done for 18-22 hours at 54° C.±1°C., shaking 600 rpm. The hybridization mixtures were then transferred toa 96 well Magnetic Separation Plate (96-well flat bottom microplate Nunc#269620). An Affymetrix Hand Held Magnetic Bead Washer (Affymetrix P/NQP0702) was used to wash the beads, thus removing all unbound materials.100 μL of 2.0 Pre-Amplifier Working Reagent (3:1000 dilution usingPreAmp solution+Amp diluent provided by manufacturer) was added to eachassay well. The Magnetic Separation Plate was sealed with adhesivebacked foil and incubated for 1 hour at 50° C.±1° C. and 600 rpm. Theunbound 2.0 Pre-Amplifier was removed and beads washed 3× with 100 μLWash Buffer (provided by manufacturer) using the handheld magneticwasher. This was followed by incubation with 2.0 Amplifier WorkingReagent, followed by Label Probe Working Reagent, and finally followedby SAPE Working Reagent (all three solutions 100 μL at 3:1000 dilution,manufacturer provided). Incubation and washing were done as describedabove. Signals from the beads were measured with a LUMINEX® FLEXMAP® 3Dinstrument (LUMINEX® Corp. Austin, Tex.), after re-suspending the beadsin 130 ul SAPE Wash Buffer, using dd gate settings of 5,000-25,000.50-100 beads per target were measured in a sample volume of 100 ul.

QUANTIGENE PLEX® 384-Well Assay Protocol:

The QUANTIGENE PLEX® 384-well Assay was optimized using technicalsupport from Affymetrix and using the 96-well assay protocol provided bythe manufacturer. 6,000 and 12,000 cells were seeded per well of a 384well plate. Proportional volumes of reagents/well were used for the384-well plate. Signal was measured as described above.

B. Results

1. Generation of iPSC-Derived RPE

A high content screening assay was developed that simultaneouslymonitors developmental and functional features of stem cell-derived RPE.FIG. 25A provides an overview of the experimental strategy for obtainingpure cultures of iPSC-derived RPE, the steps required to prepare cellsfor functional authentication and use in a high-throughput screen. iPSCswere generated from human adult female dermal fibroblasts, usingretroviral vectors expressing OCT3/4, c-MYC, SOX2, and KLF4.Pluripotency of selected iPSC colonies was determined using thefollowing three pluripotency validation assays: (I) positiveimmunostaining for pluripotency markers NANOG, SSEA4, TRA1-60, OCT3/4,c-MYC, SOX2, and KLF4 (FIG. 25B); (II) comparison of mRNA expression ofNANOG, OCT3/4, c-MYC, SOX2, and KLF4 to an embryonic stem cell line andto dermal fibroblasts. The transduced viral vector also expressedOCT3/4, c-MYC, SOX2, and KLF4, therefore, specific primer sets were usedto distinguish expression from the endogenous locus to the totalexpression (viral vector+endogenous) (FIG. 25C). Expression ofpluripotency markers from endogenous loci in iPSCs was significantlyhigher as compared to dermal fibroblasts and comparatively higher orequal to undifferentiated ES cells (FIG. 25C); (III) ability of iPSCs todifferentiate into all three germ layers. As shown in FIG. 25D, thisiPSC line differentiates into all three germ layers.

iPSCs were differentiated into RPE using the methods disclosed herein.Pigmented cell clusters with RPE-like cobblestone morphology weremanually picked and expanded in a T25 flask to obtain pure cultures ofRPE cells. Purified RPE cells were re-seeded onto transwell filters toobtain polarized confluent electrically intact RPE monolayers inapproximately 6-8 weeks Immunostaining performed at this stage forepithelial marker ZO1 confirmed the typical epithelial morphology of thecells (FIG. 26A). iPSC-derived RPE also expressed EZRIN, an apicalprocess localized protein; DCT, an enzyme important for RPEpigmentation; CLCN2, a chloride channel that is critical for volumeregulation of the RPE; and SLC16A1, an apical membrane monocarboxylatetransporter that is required for lactate transport across the RPEmonolayer and involved in intracellular pH regulation. Analysis oftransmission electron microscopy images revealed several typical RPEfeatures in these cells such as cigar and oval shaped melanosomeslocalized predominantly apically (me, arrowhead), tight junctions (tj)between adjoining cells, and apical processes (arrow) (FIG. 26B).

Many characteristic physiological responses of human native and culturedRPE cells can be measured in vitro. iPSC-derived RPE monolayer culturesdisplay several of these features. These cells are electrically intactdisplaying a transepithelial resistance across the monolayer of 170-200Ω·cm² (FIGS. 26C and 26D) and their steady-state intracellular calciumconcentration is 120 nM. Similar to native RPE, in iPSC-derived RPEstimulation of apical membrane P2Y2 receptors by ATP leads to a typicalbi-phasic response, the initial response signals the release ofintracellular calcium from the endoplasmic reticulum into the cytoplasm,and the second phase is dependent on the presence of extracellular Ca²⁺(Maminishkis et al., Investigative ophthalmology & visual science47(8):3612-3624, 2006). Application of cyclopiazonic acid blockssarco/endoplasmic reticulum Ca²⁺ ATPase, an ATP dependent calciumreuptake channel located on the ER membrane leading to steady-statecalcium increases and blunts the ATP response (FIG. 26C).Electrophysiologically, the resting membrane potentials of apical andbasal membranes are approximately −55 mV (basal side slightly moredepolarized as compared to the apical side) leading to a transepithelialpotential (TEP) across the monolayer of 2.5 mV, apical side positive(FIG. 26D). Changes in K⁺ concentration in the apical bath from 5 mM to1 mM, which mimics the in vivo alterations in subretinal space K⁺following dark to light transitions, significantly hyperpolarizes theapical membrane by 10-20 mV, thus leading to a sharp decreases in TEP(17, 20). Application of ATP to the apical membrane significantlydepolarizes the basolateral membrane following the release of calciumfrom intracellular stores and activation of basolateral membraneCa²⁺—activated Cl⁻ channels (Maminishkis et al., supra; Quinn et al.,supra; Steinberg et al., Documenta ophthalmologica. Advances inophthalmology 60(4):327-346, 1985).

The expression of pluripotency and RPE signature genes was compared inundifferentiated iPSCs, iPSC-derived RPE, and primary human fetal RPEusing qRT-PCR (Liao et al., Human molecular genetics 19(21):4229-4238,2010; Strunnikova et al., Human molecular genetics 19(12):2468-248,2010). Two major pluripotency genes OCT3/4 (also called POU5F1) and SOX2are downregulated in iPSC-derived RPE as compared to undifferentiatediPSCs suggesting a loss of the pluripotent state. However, several otherRPE-specific transcription factors, pigmentation pathways genes, visualcycle genes, structural proteins, and channels were expressed atsignificantly higher levels in iPSC-derived RPE as compared toundifferentiated iPSCs and at a lower or at similar levels as comparedto the primary RPE (Table A).

TABLE A Fold difference in gene expression in iPSC-RPE as compared to:iPSCs Primary RPE Pluripotency related transcription factors POU5F1−25.00 −1.69 KLF4 2.80 27.92 SOX2 −20.00 23.05 c-MYC −1.09 −1.78RPE-related and developmental transcription factors MITF undetermined*−5.88 TFEC undetermined* −4.17 PAX6 44.00 1.00 OTX2 4.43 −4.17 SOX994.16 −1.89 SIX3 61.01 −5.00 ROR beta 5.62 −3.57 LHX2 53.71 −10.00 CRXundetermined* −1.67 GAS1 19.60 −1.64 Pigmentation pathway genes GPNMB429.94 −5.56 MYRIP 57.92 −2.44 RAB27A 20.32 −1.96 OCA2 31.23 −5.56 DCTundetermined* −3.22 TYROSINASE undetermined* −4.00 TYRP1 undetermined*−3.03 PMEL 38.05 −10.00 Visual cycle genes ALDH1A3 27.13 −2.32 RPE65318.03 −2.94 RDH5 151.60 2.08 RLBP1 58.73 −2.86 Membrane proteins andchannels CDH1 −33.33 −25.00 CDH3 −2.86 −5.88 CLDN16 u.d. −1.23 CLDN1926.78 −3.44 TRPM1 undetermined* −1.19 TRPM3 59.88 −1.43 MERTK 2.81 −1.09BEST1 1653.15 −1.96 COL11A1 156.39 2.99 CSPG5 19.48 −3.57

These results suggest that iPSC-derived RPE, have attained an RPE-likephenotype, are but are not in a completely mature state. It isnoteworthy that despite the fact that iPSC-derived RPE express higherlevels of fetal or progenitor genes (SOX2 and KLF4, see Table A),physiological response of these cells are not significantly compromised.

A Proof-of-Principle High-Throughput Scalable Multiplex Gene ExpressionAssay Using Authenticated iPSC-derived RPE

The goals were to develop a high-throughput screening assay that cansimultaneously monitor developmental, functional, and disease markersfor RPE and perform proof-of-principle analysis of the assay. Indesigning the screening assay platform, a multiplex gene expressionassay was chosen for two main reasons: (1) it is a high content assaythat allows the simultaneous detection of genes involved in RPEdevelopment, differentiation, function, and pathology; (2) it is readilyamenable for a high-throughput screening using standard screeninginstrumentation. Genes were selected that measured different cellularprocesses—RPE development and differentiation (SOX2 and PAX6), RPEfunction (TYROSINASE, RPE65, RDH5, TRPM1, CSPG5, and BEST1), and RPEpathology (TYROSINASE, RPE65, RDH5, BEST1). FIG. 27A summarizes theoutline of the multiplex assay, FIG. 27B shows schematics of the assayprinciple, and FIG. 27C provides a list of genes used in this multiplexassay, their accession numbers, respective length of their mRNA, and theregions to which Capture Extenders and Label Extenders were hybridized.In brief, mRNA isolated in cell lysate prepared in micro-titer plates isbound to LUMINEX® beads using the anti-sense oligonucleotide technology.Detection probe (biotin-streptavidin-phycoerythrin) is hybridized tospecific mRNA also using the anti-sense oligonucleotide technology. mRNAquantification is performed using a flow cytometry based equipment thatrecognizes the specific fluorescent label on each bead and measuressignal intensity per bead.

As a proof-of-principle, the feasibility was demonstrated of a multiplexgene expression assay to simultaneously detect differential levels ofmultiple genes in a well of a 96-well or 384-well plate betweenundifferentiated iPSCs, iPSC-derived RPE, and primary human fetal RPE. Atotal of nine genes were selected for the optimization. HPRT1 and B2M,two housekeeping genes were used for data normalization, becauseprevious work suggested that the expression of these two genes does notdiffer significantly among the three cell types used in this assay (22).To analyze RPE differentiation, functioning, and pathology theexpression of SOX2, PAX6, RPE65, RDH5, CSPG5, TRPM1, and BEST1 wasmonitored. For in vitro culture, cells seeded at higher density maturefaster compared to cells seeded at lower density. Therefore, bothiPSC-derived RPE and primary fetal RPE were seeded at two celldensities, 25,000 cells/well or 50,000 cells/well (FIGS. 28B-D).Undifferentiated iPSCs were used as an additional control (FIG. 28A).iPSC-derived RPE and primary RPE were cultured for two weeks to generateconfluent pigmented monolayers. To perform the assay at the same timefor all cells types, iPSCs were cultured later and reached confluence inonly five days. A linear range of detection was calculated for eachprobe set using purified RNA, and lysates from undifferentiated iPSCs,iPSC-derived RPE (low and high cell density), and primary RPE (low andhigh cell density) (FIG. 36). All the probes detected signal above thebackground over a sixteen fold sequential dilution. The linearregression plot generated from this serial dilution generated acoefficient of determination value of more than 0.97 in all cases,indicating that the probes were able to detect a signal over a largerange of mRNA concentrations.

The results of this assay are presented as fold change in geneexpression in iPSC-derived RPE at two different cell densities ascompared to undifferentiated iPSCs (FIGS. 28E and 28F) and as comparedto primary fetal RPE at high cell density (FIGS. 28G and 28H). Asexpected, compared to undifferentiated iPSC, iPSC-derived RPE expresseslower levels of neural progenitor factor SOX2 and much higher levels ofRPE-specific genes PAX6, RPE65, RDH5, TRPM1, and BEST1. CSPG5 anextracellular protein is expressed at similar levels in the two celltypes. An important goal of this optimization was to compare resultsobtained from the multiplex assay to those obtained from a standardqRT-PCR assay (Table A). Therefore, the Pearson's correlationcoefficient (r) was determined between fold change in gene expressionobtained from the multiplex assay and from the qRT-PCR assay. Theresults show that the two assays are significantly correlated(r-value=0.69 and 0.75). This provides confidence that the multiplexassay measures relevant gene expression changes and can be used for ahigh-throughput screen. In conclusion, this high-content gene expressionassay can be performed in one and a half days in a high-throughputfashion on undifferentiated iPSCs, iPSC-derived RPE, and primary humanRPE. When compared to primary fetal RPE, iPSC-derived RPE express lowerlevels of differentiation RPE markers (RPE65, RDH5, CSPG5, BEST1) andhigher levels of progenitor transcription factors PAX6 and SOX2,suggesting a relatively immature state of these cells. It is, however,important to note that the results obtained by comparison of primary RPEand iPSC-derived RPE in multiplex assay are strongly correlated with thestandard qRT-PCR assay presented in Table A.

Evaluating the Limit of Detection and Increasing the Throughput of theAssay

The limit of detection for each transcript in a given assay well wastested, another important aspect of the multiplex assay. The suggestedminimal limit of detection for the multiplex gene expression assay isless than or equal to 200 transcripts when analyzing one target per wellof a 96-well plate. The following formula was used to calculate thelimit of detection (LOD) of our probe set: LOD=Average background valueof four wells+3× standard deviation of the average background value(Affymetrix, manufacturer manual). The assay was significantly moresensitive than the described limit, and the following values ofdetection were obtained: SOX2=41, PAX6=32, RPE65=24, RDH5=10, CSPG5=19,TRPM1=25, BEST1=11, HPRT1=55, and B2M=30. In view of these transcriptnumbers, the assay was tested in a 384-well format for increasedscreening throughput and reduce reagent and screening costs.

To test the linearity of the range of signal detection, the assay wasperformed using two cell densities, 6,000 and 12,000 cells/well in384-well plate. As expected, after two weeks of culture in 384-wellplates, the lower cell density produced less confluent and lesspigmented cell layer both for primary fetal RPE cells and foriPSC-derived RPE (FIG. 35A-35D). Two different bead concentrations—700beads/well per probe, as recommended for a 96-well plate, and 375beads/well per probe, were tested. An additional probe, TYROSINASErepresenting RPE maturity was included in this assay. FIG. 35 shows thatthe assay was successfully optimized in 384-well plate using both beadconcentrations. This data support the following three conclusions: (1)results obtained in 384-well plates are similar to those obtained in96-well plates. For example, when compared to primary RPE, iPSC-derivedRPE express higher levels of SOX2 and PAX6 and lower levels of RPEdifferentiation/functioning genes; (2) the two bead concentrations givealmost identical results in the 384-well plate format; (3) the multiplexassay in 384-well plates correlates better (Pearson's correlationcoefficient of 0.91 and 0.89 respectively for high and low beads) withthe qRT-PCR data as compared to the 96-well plate assay. In conclusion,the present results demonstrate that changes in expression of up to tendifferent genes can be measured simultaneously using iPSC-derived RPE384-well plates.

The approach presented here provides a well-defined method foridentifying small molecules that downregulate the expression ofprogenitor genes and upregulate the expression of differentiation genes,thus allowing systematic maturation of pluripotent stem cell-derived RPEin culture. This iPSC-derived RPE can be more effective in therapeuticintervention of macular degenerative diseases and also serve aseffective disease model. This screening method also can be used toidentify small molecules that alter the activity of putativedisease-causing pathways and lead to the discovery of therapeutic drugs.

Example 9 QUANTIGENEPLEX®2.0 Reagent System—96-Well Assay Protocol

The QUANTIGENE PLEX® 96-well Assay was done using following protocol.Cells were seeded per well in 96 well plate and let adjust to the wellsfor 10 days at 37° C., 5% CO₂, with manual media exchanges every otherday. Cells from individual wells were then lysed in 200 μL working lysismixture (Affymetrix) for 30 minutes at 50° C. 80 μL of cell lysate weretransferred to the assay's hybridization plate (96-well clearpolypropylene plate Abgene #AB0796), where each well already contained20 μL of Working Bead Mix. Each 20 μL of Working Bead Mix contained 6.6μL of Lysis Mixture, 5.2 μL of nuclease free water, 0.2 μL of ProteinaseK solution, 2 μL of Blocking Reagent, 5 μL of Probe Set, 1 μL ofmagnetic Luminex beads (1000 beads per gene/well). The Probe Set andLuminex Beads correspond to QuantiGene Plex Set-Panel #11828. Thehybridization plate was sealed with the supplied clear,pressure-activated seals and placed in a heating/shaking incubator(LabNet VorTemp 56 P/N S-0256-Q) and incubated for 18-22 hours at 54°C.±1° C. and shaking at 600 rpm. Each of the Magnetic beads used in themultiplex assay have different ssDNA Capture Probes, that work togetherwith the probe set to capture different mRNA targets onto differentbeads. The overnight hybridization mixtures were then transferred to a96 well Magnetic Separation Plate (96-well flat bottom microplate Nunc#269620). An Affymetrix Hand Held Magnetic Bead Washer (Affymetrix P/NQP0702) was used to wash the beads, thus removing all unbound materials.100 μL of 2.0 Pre-Amplifier Working Reagent (3:1000 dilution usingPreAmp solution+Amp diluent provided by manufacturer) was added to eachassay well. The Magnetic Separation Plate was sealed with adhesivebacked foil and incubated for 1 hour at 50° C.±1° C. and 600 rpm. Theunbound 2.0 Pre-Amplifier was removed and beads washed 3× with 100 μL ofWash Buffer (provided by manufacturer) using the handheld magneticplate. 100 μL of 2.0 Amplifier Working Reagent (3:1000 dilution usingAmp solution+Amp diluent provided by manufacturer) was added to eachassay well. The Magnetic Separation Plate was sealed and incubated for 1hour at 50° C.±1° C. and 600 rpm. The unbound 2.0 Amplifier was removedand beads washed 3× with 100 μL Wash Buffer using the handheld magneticbead washer. 100 μL of Label Probe Working Reagent (3:1000 dilutionusing LabelProbe solution+LabelProbe diluent provided by manufacturer)was added to each assay well. The Magnetic Separation Plate was sealedand incubated for 1 hour at 50° C.±1° C. and 600 rpm. The unbound LabelProbe was removed and beads washed 3× with 100 μL Wash Buffer using thehandheld magnetic bead washer. 100 μL of SAPE Working Reagent (3:1000dilution using SAPE solution+SAPE diluent provided by manufacturer) wasadded to each assay well. The Magnetic Separation Plate was sealed,wrapped in aluminum foil, and incubated for 30 min at RT and 600 rpm.The unbound SAPE Working Reagent was removed and beads washed 3× with100 μL SAPE diluent using the handheld magnetic bead washer. Signal frombeads was measured with a Luminex Flex Map 3D instrument after firstre-suspending the beads in 130 μL of SAPE diluent, using DD gatesettings between 50-100 beads per message, a sample volume of 50 μL, anda sample timeout of 45 seconds.

Example 10 Aphidicolin

Aphidicolin is a reversible inhibitor of eukaryotic nuclear DNAreplication that blocks the cell cycle at early S phase. Its structureis that of a tetracyclic small molecule. Aphidicolin added to iPS cellderived RPE growing on artificial membranes/scaffolds, improves theirRPE phenotype. It causes committed RPE cells to further mature and formfully-differentiated and highly polarized tissue monolayers (see FIGS.30-31). Aphidicolin is dissolved in fourth medium and RPE cells arecultured in it for 6-8 weeks before analysis is done.

Fully-differentiated and polarized RPE tissues on scaffolds will providemore effective therapies for retinal degenerative diseases and will alsoprovide more “natural” models for in vitro disease modeling and for highthroughput screenings. Aphidicolin treatment generates tissues withfully mature tight junctions and more mature physiological responses.When combined with a biodegradable scaffold, this compound stimulatesRPE monolayer to secrete its own extracellular matrix and attain a realtissue like features.

Example 11 Alginate Coating Enhances Cell Viability and Reduces CellDetachment

An alginate-enhanced Cryopreservation Protocol is provided below. Theresults are shown in FIG. 32A-32D.

-   -   1. Use 10 mm biopsy punch (ACCU-PUNCH®, USA) to cut out        transwell membrane with confluent monolayer of hfRPE.    -   2. Dip sample into autoclave-sterilized 0.7% sodium alginate        (Sigma Aldrich, United Kingdom) dissolved ddH₂O for 0.5 seconds.    -   3. Dip sample into autoclave-sterilized 2% CaCl₂ (Sigma Aldrich,        USA) in ddH₂O for 0.5 seconds, to polymerize alginate.    -   4 Immediately place sample into 2 mL cryovial (Corning, USA)        containing either 1 mL CryoStor CS10 (STEMCELL®, Canada) or 10%        DMSO cryopreservation media (500 μL 5% RPE, 400 μL FBS, 100 μL        DMSO).    -   5. Place cryovials on ice for 30 minutes.    -   6. Transfer cryovials to a Mr. Frosty freezing container (Thermo        Scientific, USA), pre-cooled to 4° C., and cool to −80° C. at a        rate of 1° C./min.    -   7. Recover cryopreserved tissue by partially melting samples in        a 37° C. water bath.    -   8. Add 1 mL pre-warmed 5% RPE media to cryovial to complete        melting.    -   9. Transfer sample into 1 mL 5% RPE to rinse away        cryopreservation media components.    -   10. Move sample into 1 mL 5% RPE medium.    -   11. Allow recovery for 3 days, changing media daily.    -   12. Test cell viability using ethidium homodimer-1 (2.5 μM).

Prepare the SNAPWELL™ (Costar, US) System (FIG. 33A-33E)

-   -   1. Manually separate the well top from the well bottom (FIG. 2A)    -   2. Place the scaffold (Stellenbosch Nanofiber Company (Pty)        Ltd., South Africa) into the center of the SNAPWELL™ bottom so        that it lays flat (FIG. 2B).    -   3. Place an O-ring (polytetrafluoroethylene (PTFE), 12 mm×10.25        mm×1.2 mm, Superior Washer and Gasket Corp, US) on top of the        scaffold so that it holds the scaffold in place. The O-ring must        be previously sterilized by 20-minute treatment in 70% ethanol        (FIG. 2C).    -   4. Place the SNAPWELL™ top on top of the O-ring and gently press        into place (FIG. 2D).

Prepare the Scaffold for Cell Seeding

-   -   1. Dispense 200 mL of 1×PBS (Life technologies, US) into the        SnapWell™ with the scaffold. Leave for two hours at room        temperature.    -   2. Aspirate out the PBS and add 200 mL of human extracellular        matrix (ECM)/1×HBSS with phenol red (PR) solution to each        SnapWell™ (1 vial ECM: 20 mL HBSS). Human ECM (BD BioSciences,        US 1×HBSS with phenol red (Life technologies, US).    -   3. Place the plate, lid off, under UV light for 2 hours.    -   4. After 2 hours, turn off UV. To prevent the solution from        drying out during cell preparation add 100 mL 1×HBSS without PR        (Life technologies, US) to the 200 mL of human ECM/1×HBSS with        PR solution.

Seed Cells

-   -   1. Aspirate out the 300 mL of human ECM/1×HBSS solution.    -   2. Dispense 500 mL of cell suspension (in 5% RPE media at a        density of 250,000 cells per mL) into each well.

The use of induced pluripotent stem (iPS) cell technology todifferentiate RPE provides an autologous or allogeneic cell-basedtherapy for patients with ocular degeneration. RPE cells derived fromstem cells have been injected as a cell suspension into the eyes ofpatients with retinal degeneration. RPE tissue was generated from iPSCderived RPE grown as a polarized confluent monolayer on a biodegradablescaffold. By transplanting a single monolayer tissue, one cancircumnavigate the problems associated with suspensions.

RPE cells were cultured on biocompatible membranes, such as parylene-Cand polyimide (Diniz et al, IOVS 2013). These membranes do not degradeovertime, leaving a permanent foreign entity in the eye, which over timewill get clogged with proteinaceous material. Disclosed herein is atechnique that allows hydrolytic degradation to remove the scaffold asthe RPE layer matures and creates its own external environment.Therefore, our method for creating functional and polarized RPEmonolayers on scaffolds is a viable tissue engineering strategy thatprovides therapy for retinal degenerative diseases.

A protocol was produced that utilizes biodegradable scaffolds forculturing RPE cells into a functional tissue that can be translated intoin vivo models to combat rental diseases. The main components of thismethod are a CORNING® COSTAR® SNAPWELL™ plate, a bioinert 0-ring, and abiodegradable scaffold. SNAPWELL™ plates provide the structure andplatform for the biodegradable scaffolds. The microporous membrane thatcreates an apical and basal side is ideal for providing support to thescaffold as well as isolating the distinct sides of the polarized layerof cells. The ability of the SNAPWELL™ insert to detach the membraneallows the support ring of the insert to be used an anchor for thescaffold. However, after combing the support ring and membrane, there isa small void between the bottom lip of the support ring and the porousmembrane itself. Therefore, we have utilized a bioinert 0-ring to fitperfectly and fill the void. Now, the support rig can apply pressureuniformly around the outer edge of the membrane to hold the scaffoldagainst the membrane.

First, the SNAPWELL™ apparatus was disassembled, the support ring wasremoved from the bottom membrane. Next, the biodegradable scaffold, wasprecut to match the interior dimensions of the SNAPWELL™ membrane, andwas placed flat along the bottom of the membrane. The 0-ring was thenplaced directly into the membrane portion of the SNAPWELL™ insert on topof both the scaffold and the membrane. Then, the SnapWell™ insert wasreassembled, ensuring that the support ring presses on the 0-ring sothat there is no gap between the support rig and the porous membrane.

Prior to adding any solution to the culturing device, the scaffolds werewetted and sterilized using techniques such as ethanol treatments andultraviolet light exposure. This scaffold and membrane structure wasthen suitable for culturing polarized cells by seeding cells on top ofscaffold. After the cells were placed onto the scaffold, the cultureconditions were maintained including applying additional factors untilthe tissue is fully develop and functional. The tissue was then readyfor use in transplantation to in vivo models or other in vitro studies.

The culturing assembly can be disassembled, detaching the support ringfrom the membrane and lifting the 0-ring out of the membrane ring. Thetissue could also be directly punched or cut out of the device. Sincethere is nothing chemically holding the tissue to the porous membrane,the tissue lifts off and away from the membrane after being cut.Resulting RPE tissue can be transplanted as a single entity into thesubretinal space of patients with retinal degeneration in order toresurrect the degrading areas by preventing photoreceptor death andpromoting photoreceptor regeneration.

In view of the many possible embodiments to which the principles of ourinvention may be applied, it should be recognized that illustratedembodiments are only examples of the invention and should not beconsidered a limitation on the scope of the invention. Rather, the scopeof the invention is defined by the following claims. We therefore claimas our invention all that comes within the scope and spirit of theseclaims.

We claim:
 1. A method for producing human retinal pigment epithelial(RPE) cells, comprising: (a) culturing human induced pluripotent stemcells (hiPSCs) in a human embryonic stem cell culture medium comprisinghuman basic fibroblast growth factor (bFGF) and not containingingredients obtained from non-human animals to produce small embryoidbodies of 200-500 cells; (b) culturing the small embryoid bodies fromstep (a) in a first medium comprising retinal induction medium (RIM) androck inhibitor (RI), wherein the RIM comprises Wnt inhibitor CK1-7, aNodal pathway inhibitor, Noggin, and 1 to 3% v/v knockout serumreplacement, wherein the Nodal pathway inhibitor is SB-431542 orSB-505124, to form embryoid bodies that have increased efficiency of RPEdifferentiation; (c) culturing the embryoid bodies that have increasedefficiency of RPE differentiation from step (b) on a matrigel coatedtissue culture substrate-in a second medium comprising a retinaldifferentiation medium that does not comprise basic fibroblast growthfactor (bFGF) and comprises Dickkopf-related protein 1 (DKK1), CK1-7, abFGF inhibitor, and Noggin, wherein the bFGF inhibitor is PD0325901,PD98059, PD161570, or PD166285, to form differentiating RPE cells thatexpress PAX6 and MITF; (d) culturing the differentiating RPE cells fromstep (C) in a third medium comprising retinal media comprising Activin Aand Wnt3a to produce cells that have increased expression of MITF andPAX6 and increased RPE differentiation efficiency; and (e) culturing thecells that have increased expression of MITF and PAX6 and increased RPEdifferentiation efficiency from step (d) in a fourth medium comprising aRPE cell medium comprising a non-canonical Wnt 5a inducer, DKK1, SU5402,and cyclopamine to produce human RPE cells that express TYR, TYRP1,MYRIP, Cadherin 1 or Cadherin 3 and TRPMI 1 or TRPMI 3, therebyproducing human RPE cells.
 2. The method of claim 1, wherein the firstmedium comprises 3.5 to 9 mM of CKI-7.
 3. The method of claim 1, whereinthe Nodal pathway inhibitor is SB-431542.
 4. The method of claim 3,wherein the first medium comprises 3.5 to 9 mM of SB-431542.
 5. Themethod of claim 1, wherein the retinal induction medium does notcomprise serum.
 6. The method of claim 1, wherein the inhibitor of bFGFis PD032590.
 7. The method of claim 6, wherein the second mediumcomprises 0.5 to 2 mM of PD0325901.
 8. The method of claim 1, whereinthe second medium comprises 3.5 to 9 mM of CK1-7 and 3.5 to 9 mM ofSB-431542.
 9. The method of claim 1, wherein the first medium comprises50 ng/ml of Dickkopf-related protein 1 (DKK1).
 10. The method of claim1, wherein the third medium comprises 100 to 200 ng/ml of Activin A. 11.The method of claim 1, wherein the third medium comprises 75 to 150ng/ml of Wnt3a.
 12. The method of claim 1, wherein the second mediumcomprises 75 to 150 ng/ml of DKK1.
 13. The method of claim 1, whereinthe fourth medium comprises 75 to 150 ng/ml of WNT5a, 75 to 150 ng/ml ofDKK1, 5 μM Cycolopamine and 10 μM of SU5402.
 14. The method of claim 1,wherein step (a) comprises culturing the cells in the presence of 1.5%v/v knock out serum replacement.
 15. The method of claim 1, wherein thetissue culture substrate is a transwell plate.
 16. The method of claim1, further comprising expressing OCT4, SOX2, LIN28 and Nanog in a humanfetal RPE cell to produce the human induced pluripotent stem cells. 17.The method of claim 1, wherein the human induced pluripotent stem cellscomprise a nucleic acid encoding a marker operably linked to a RPE cellspecific promoter.
 18. The method of claim 17, wherein the retinalspecific promoter is a tyrosinase promoter.
 19. The method of claim 1,wherein the embryoid bodies are cultured in the first medium for 48hours.
 20. The method of claim 1 wherein the embryoid bodies arecultured in the second medium for 18 to 24 days.
 21. The method of claim20, wherein the embryoid bodies are cultured in the second medium forthree weeks.
 22. The method of claim 1, wherein the differentiating RPEcells are cultured in the third medium for 18 to 24 days.
 23. The methodof claim 22, wherein the differentiating RPE cells are cultured in thethird medium for three weeks.
 24. The method of claim 1, wherein thecells are cultured in the fourth medium for 12 to 16 days.
 25. Themethod of claim 24, wherein the cells are cultured in the fourth mediumfor two weeks.
 26. The method of claim 1, further comprising maintainingthe RPE cells in the fourth medium and 5% v/v fetal serum.
 27. Themethod of claim 26, comprising maintaining the RPE cells in the fourthmedium and 5% v/v fetal serum for six to eight weeks.